Methods and compositions for the diagnosis of ovarian cancer

ABSTRACT

A diagnostic reagent or device comprises at least one ligand capable of specifically complexing with, binding to, or quantitatively detecting or identifying the biomarker chloride intracellular channel protein 4 (CLIC4) or an isoform, pro-form, modified molecular form including posttranslational modification, or unique peptide fragment or nucleic acid fragment thereof. An alternative diagnostic reagent or device comprises ligand or ligands capable of specifically complexing with, binding to, or quantitatively detecting or identifying multiple tropomyosin biomarkers. Optionally, such reagent or device includes a signaling molecule and/or a substrate on which the ligand is immobilized. Other reagents and methods of diagnosing ovarian cancer include use of CLIC4 ligands and/or multiple tropomyosin ligands with an additional ovarian cancer biomarker. For example, CLIC4 combined with one or more of CLIC1 and/or one or multiple members of the tropomyosin family, e.g., TPM1, TPM2, TPM3 or TPM4, and further optionally including CTSD-30 kDa and/or PRDX-6, among other ovarian cancer biomarkers can form a characteristic diagnostic pattern or profile of expression that is diagnostic of the disease. Still other embodiments are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the priority of U.S. ProvisionalPatent Application No. 61/709,695, filed Oct. 4, 2012, which applicationis incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.CA131582 and CA10815, awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancerin the United States and the fifth-leading cause of cancer-related deathin women in the United States. An estimated 22,280 new cases weredetected and 15,500 deaths occurred in the US in 2012. When diagnosedearly (Stages I/II), treatment is generally successful, with a five-yearsurvival rate of up to 90%. Unfortunately, most cases are not detecteduntil after the cancer has spread, resulting in a dismal five-yearsurvival rate for patients with advanced disease (stages III and IV) of30% or less. The high mortality rate of ovarian cancer is due largely tothe lack of effective screening tests for early detection or diagnosisof EOC.

Current screening methods for ovarian cancer typically use a combinationof pelvic examination, transvaginal ultrasonography, and assays forprotein biomarkers, such as serum cancer antigen 125 (CA-125). CA125 isrecognized as a poor protein biomarker for early detection due to itshigh false positive rate and poor sensitivity and specificity. Assaysfor human epididymis protein-4 (HE4), or multivariate OVA1 are onlyapproved for monitoring disease recurrence, therapeutic response, or foruse in managing women with an ovarian adnexal mass.

A recently completed study comparing many of these protein biomarkersshowed that none of them performed better than CA125 as a biomarker forovarian cancer. A few groups also have used panels of biomarkers andobtained better sensitivity and specificity than CA125 alone when usedin diagnostic samples. However, a recent study found that availablebiomarker panels did not outperform CA125 when used in prediagnosticsamples.

Compositions and methods are urgently needed to diagnose early-stage EOCwith high sensitivity and specificity and for clinical management of thedisease after initial diagnosis.

SUMMARY OF THE INVENTION

In one aspect, a diagnostic reagent or device comprises the biomarkerchloride intracellular channel protein 4 (CLIC4) or an isoform,pro-form, modified molecular form, or unique peptide fragment or nucleicacid fragment thereof. In another aspect, a diagnostic reagent or devicecomprises a ligand capable of specifically complexing with, binding to,or quantitatively detecting or identifying the biomarker chlorideintracellular channel protein 4 (CLIC4) or an isoform, pro-form,modified molecular form, or unique peptide fragment or nucleic acidfragment thereof. In certain embodiments, the biomarker sequence orligand in the reagent or device is associated with a molecule or moietycapable alone or in combination with one or more additional molecules ofgenerating a detectable signal. In other embodiments, the biomarkersequence or ligand in the reagent or device is associated with asubstrate on which the sequence or ligand is immobilized.

In another aspect, a diagnostic reagent or device comprises one or moretropomyosin proteins, e.g., TPM1, TPM2, TPM3, TPM4, or a common orshared sequence formed of two or more TPM proteins or an isoform,pro-form, modified molecular form, unique peptide fragment or nucleicacid fragment thereof, or proteins in the same biomarker family orexpressed from a related gene, having at least 20% sequence homology orsequence identity with any tropomyosin biomarker. In another aspect, adiagnostic reagent or device comprises a ligand capable of specificallycomplexing with, binding to, or quantitatively detecting one or moretropomyosin proteins, e.g., TPM1, TPM2, TPM3, TPM4, or a common orshared sequence in two or more TPM proteins or an isoform, pro-form,modified molecular form, or unique peptide fragment or nucleic acidfragment thereof or proteins in the same biomarker family or expressedfrom a related gene, having at least 20% sequence homology or sequenceidentity with any tropomyosin biomarker. In certain embodiments, thebiomarker sequences or ligand(s) in the reagent or device are associatedwith a molecule or moiety capable alone or in combination with one ormore additional molecules of generating a detectable signal. In otherembodiments, the biomarker sequences or ligand(s) in the reagent ordevice are associated with a substrate on which the ligand isimmobilized.

In still a further aspect, the diagnostic reagent or device comprises aset of multiple biomarkers or multiple ligands to biomarkers, eachligand individually capable of specifically complexing with, binding to,or quantitatively detecting or identifying a single biomarker. In oneembodiment of such diagnostic reagents or devices, one requiredbiomarker is CLIC4. In another embodiment of such diagnostic reagents ordevices, required biomarkers are one or more tropomyosin proteins, e.g.,TPM1, TPM2, TPM3, TPM4, or a common or shared sequence in two or moreTPM proteins, as defined herein.

In still another aspect, the diagnostic reagent or devices describedabove further comprise a set of multiple biomarkers or multiple ligands,each ligand individually capable of specifically complexing with,binding to, or quantitatively detecting or identifying a singlebiomarker, depending upon whether the reagent is formed of biomarkersequences or ligands to biomarker sequences. The additional biomarker isone that indicates the presence of ovarian cancer in a human subject.The biomarker sequences or fragments are derived from the selectedadditional biomarker. Each additional ligand is capable of specificallycomplexing with, binding to, or quantitatively detecting, or identifyingthe additional biomarker. In one embodiment, the additional marker ischloride intracellular channel protein 1 (CLIC1) or an isoform,pro-form, modified molecular form, or unique peptide fragment or uniquenucleic acid fragment of the additional biomarker or proteins in thesame biomarker family or expressed from a related gene, having at least20% sequence homology or sequence identity with any tropomyosinbiomarker.

In one embodiment in which at least a required biomarker is CLIC4, theadditional biomarker is one or more tropomyosin proteins, e.g., TPM1,TPM2, TPM3, TPM4, or a common or shared sequence within two or more TPMproteins, or an isoform, pro-form, modified molecular form, or uniquepeptide fragment or unique nucleic acid fragment thereof, proteins inthe same biomarker family or expressed from a related gene, having atleast 20% sequence homology or sequence identity with any tropomyosinbiomarker. In another embodiment, the biomarkers are CLIC4 and at leastone or more additional biomarkers selected from one or multipletropomyosin biomarker proteins 1 through 4, and/or CLIC1. Stilladditional ovarian cancer biomarkers can form part of the set ofmultiple markers.

In yet another aspect, a kit, panel or microarray comprising at leastone diagnostic reagent with is a biomarker CLIC4 and/or one or moretropomyosins, or ligands capable of binding thereto, and optionally oneor more reagents that are different ovarian cancer biomarkers discussedherein or ligands capable of binding thereto.

In a further aspect, a method for diagnosing or detecting or monitoringthe progress of ovarian cancer in a subject is provided. In oneembodiment, the method comprises contacting a sample obtained from atest subject with a diagnostic reagent or device comprising a ligandcapable of specifically complexing with, binding to, or quantitativelydetecting or identifying the biomarker chloride intracellular channelprotein 4 (CLIC4) or an isoform, pro-form, modified molecular form, orunique peptide fragment or nucleic acid fragment thereof. The proteinlevels of CLIC4 (or peptides of CLIC4 serving as surrogates of the CLIC4protein) are then detected or measured in the sample or from a proteinlevel profile generated from the sample. The protein levels of the CLIC4biomarker in the subject's are compared with the level of the samebiomarker in a reference standard. A significant change in protein levelof the subject's sample from that in the reference standard indicates adiagnosis, risk, or the status of progression or remission of ovariancancer in the subject. In one embodiment of this method, an additionalstep involves detecting or measuring in the sample or from a proteinlevel profile generated from the sample, the protein levels of one ormore additional ovarian cancer biomarkers; and comparing the proteinlevels of the CLIC4 biomarker in relation to the levels of theadditional biomarkers in the subject's sample with the same biomarkersin a reference standard or profile. Analogous methods are provided forother biomarkers or biomarker sets described herein.

In another aspect, use of any of the diagnostic reagents describedherein in a method for the diagnosis of ovarian cancer is provided.

Other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a quantitation of the biomarker tropomyosin 1 (TPM1)extracted from an LC-MS chromatogram using label-free discovery mode,and GeLC-MS/MS analysis of patient serum pools. Intensities ofidentified peptides unique to this biomarker were summed for each offour serum pools (B-benign disease; C1, C2, C3—three different advancedovarian cancer pools. LC-MS data from corresponding gel fractions ofdifferent serum pools were aligned and quantitatively compared usingElucidator software.

FIG. 1B is a quantitation as described in FIG. 1A for the biomarkertropomyosin 2 (TPM2), expressed from a different gene than thatexpressing TPM1.

FIG. 1C is a quantitation as described in FIG. 1A for the biomarkertropomyosin 3 (TPM3), expressed from a different gene than thatexpressing TPM1.

FIG. 1D is a quantitation as described in FIG. 1A for the biomarkertropomyosin 4 (TPM4), expressed from a different gene than thatexpressing TPM1.

FIG. 1E is a quantitation as described in FIG. 1A for the biomarkerchloride ion channel protein 1 (CLIC1).

FIG. 1F is a quantitation as described in FIG. 1A for the biomarkerchloride ion channel protein 4 (CLIC4) expressed from a different genethan that expressing CLIC1.

FIG. 2A is a sequence alignment of CLIC4 human (SwissProt Ref: Q9Y696;SEQ ID NO: 1) and 99% identical mouse (SwissProt Ref: Q9QYB1; SEQ ID NO:2) CLIC4 sequence.

FIG. 2B is a sequence alignment of four human CLIC biomarkers expressedfrom different genes in the CLIC family. These biomarker gene productsare of similar size and located in the 25-50 kDa region (CLIC1—SwissProtRef: 000299; SEQ ID NO: 3; CLIC2—SwissProt Ref: 015247; SEQ ID NO: 4;CLIC3—SwissProt Ref: 095833; SEQ ID NO: 5; and CLIC4—SwissProt Ref:Q9Y696; SEQ ID NO: 1). Database identifiers are from the Swiss-Protdatabase. Tryptic sites (K or R) are indicated in bold—underlinedsequences—peptides identified in the xenograft mouse serum. Greyhighlight—peptides identified only in the patient serum pools. Lowercase sequences—peptides identified in both the xenograft mouse andpatient serum. Boxed sequences—peptides used for MRM quantitation.

FIG. 3 is a sequence alignment of selected isoforms of the biomarkerTPM1, i.e., TPM1 isoform 5 SEQ ID NO: 6, TPM1 isoform 6 SEQ ID NO: 7,TPM1 variant SEQ ID NO: 8, TPM1 isoform CRA_i SEQ ID NO: 9, TPM1 variant6 SEQ ID NO: 10, and TPM1 cDNA FLJ55130 SEQ ID NO: 11. TPM1 isoformsthat contain all five xenograft-identified peptides (lower casesequences) were aligned using the Clustal algorithm. Two additionalpeptides (grey highlight) were subsequently identified in analyses ofovarian cancer patient sample pools. Inclusion of these peptidessuggests the presence of TPM1 Q1ZYL5 or B7Z596. The database type (SP,Swiss-Prot entry; TR, TrEMBL entry), identifier, and brief descriptionare indicated for each sequence. Tryptic sites (K or R) are indicated inbold. Boxed sequences—peptides used for MRM quantitation.

FIG. 4 are GeLC-MRM quantitations of tropomyosin biomarkers inindividuals without ovarian cancer (WCS), individuals with benign (B),and individuals with late-stage (T) ovarian cancer. The followingpeptides were used to derive these values:

TABLE 1 Peptides represented in FIG. 4 Biomarker Peptide SEQ ID NO:TPM1 variant 6 ETAEADVASLNR AA43-54 of SEQ ID NO: 10 SLQEQADAAEERAA16-27 of SEQ ID NO: 10 LVIIESDLER AA133-142 of SEQ ID NO: 10 AELSEGQVRAA147-155 of SEQ ID NO: 10 TPM3 IQVLQQQADDAEER 12 TPM4 AEGDVAALNR 13IQALQQQADEAEDR 14 [K]LVILEGELER 15 TPM (common) [R]IQLVEEELDR 16

FIG. 5A is a scatter plot showing GeLC-MRM quantitation of the CLIC1biomarker in serum of normal (n=6), benign (n=9), and ovarian cancerpatients (15 Stage III, 3 Stage IV). P-values were calculated using theMann-Whitney test with Bonferroni adjustment. Horizontal bars in eachgroup indicate the median serum level of the protein. Peptidesassociated with this biomarker are shown in Table 5.

FIG. 5B is a scatter plot showing GeLC-MRM quantitation of the CLIC4biomarker as described in FIG. 5A. Peptides associated with thisbiomarker are shown in Table 4.

FIG. 5C is a scatter plot showing GeLC-MRM quantitation of the TPM1,variant 6 biomarker as described in FIG. 5A. Peptides associated withthis biomarker are shown in Table 6.

FIG. 5D is a scatter plot showing GeLC-MRM quantitation of the TPM3biomarker as described in FIG. 5A. Peptides associated with thisbiomarker are shown in Table 7.

FIG. 5E is a scatter plot showing GeLC-MRM quantitation of the TPM4biomarker as described in FIG. 5A. Peptides associated with thisbiomarker are shown in Table 7.

FIG. 5F is a scatter plot showing GeLC-MRM quantitation of the TPMfamily of biomarkers as described in FIG. 5A. The peptide common to allfour gene products and used for this quantitation is shown in Table 8.

FIG. 6A is a ROC curve of the CLIC1 biomarker generated from Control (6normal, 9 benign) and Cancer (15 Stage III, 3 Stage IV) datasets. Thearea under the ROC curve is indicated for each biomarker. The peptidesassociated with each biomarker are shown in Tables 4-8.

FIG. 6B is a ROC curve of the CLIC4 biomarker, as described in FIG. 6A.

FIG. 6C is a ROC curve of the TPM1, variant 6 biomarker, as described inFIG. 6A.

FIG. 6D is a ROC curve of the TPM3 biomarker, as described in FIG. 6A.

FIG. 6E is a ROC curve of the TPM4 biomarker, as described in FIG. 6A.

FIG. 6F is a ROC curve of the TPM family, as described in FIG. 6A.

DETAILED DESCRIPTION OF THE INVENTION

New serological biomarkers for early detection and clinical managementof ovarian cancer are urgently needed, and many candidates have beenreported. A major challenge frequently encountered when validatingcandidates in patients is establishing quantitative assays thatdistinguish between highly homologous proteins. It is important todifferentiate between protein isoforms as well as related proteinmembers of a family because these sequence differences are oftenassociated with a vital distinct role that is critical to the proteinstructure or function. In this regard, any assay, including sandwichELISA assays, could give misleading results if the biomarker specificityis unknown or if multiple related biomarker proteins are quantitated asa group.

The present inventors developed a gel-based, label-free MRM quantitationapproach (GeLC-MRM) as a rapid, first-level biomarker verificationstrategy using human plasma or serum samples. As disclosed in theexamples below, the inventors used in-depth GeLC-MS/MS analysis ofpatient serum pools and isoform-specific MRM assays (Tang et al, 2012,cited above; Tang, H. Y et al, J Proteome Res 2011, 10, (9), 4005-17;and Beer, L. A et al, J Proteome Res 2011, 10, (3), 1126-38) to identifyand quantitate additional EOC biomarkers. Related proteins in twoprotein families were detected that significantly distinguished betweencancer and control patients to a high sensitivity.

The inventors were able to use quantitative MRM assays to distinguishbetween all related protein members and the isoforms of certain membersthat were detectable in low abundance in pools of ovarian cancer patientsera. By using a combination of unique and shared peptides together withcorrelation and factor analysis, the inventors determined that suchunidentified related proteins either do not occur at a significant levelor change in parallel with the proteins explicitly defined by uniquepeptides. In summary, the inventors found that the CLIC4 biomarker andat least four tropomyosins biomarker proteins (TPM 1 variant 6, TPM2,TPM3, and TPM4) are significantly elevated in ovarian cancer patientscompared with non-cancer controls. These proteins, as well as thepreviously identified biomarkers CLIC1, PRDX6, and CTSD, among othersidentified in PCT/US2012/54136, are promising new EOC biomarkers fordistinguishing between patient cohorts and diagnosing ovarian cancerfrom non-cancer controls. As such, they form the basis of noveldiagnostic reagents and devices, as well as methods for diagnosing ordetecting the existence or absence of, or monitoring the progress of,ovarian cancer in a subject. Such compositions and method use one ormore of the biomarkers, e.g., CLIC4 in optional combination with one ormore other ovarian cancer-associated biomarkers, or CLIC4 in combinationwith multiple related TPM proteins, to diagnose and monitor theprogression and treatment of EOC.

Protein abundance levels of biomarkers in blood, in some embodiments,are dependent upon expression levels in tissues of origin (e.g., ovariantumors), as well as rate of shedding into the blood and rate ofclearance from the blood. While increased expression in a tumor oftenwill correlate with increased abundance levels being observed in theblood, this is not necessarily always true. Therefore, the methods andcompositions in one aspect refer to compositions that detect proteinbiomarkers and to protein assay methods. However, one of skill in theart, given the teachings contained herein, would readily understand thatnucleic acid expression levels of the biomarkers and reagents andmethods for their detections may be similarly practiced, without undueexperimentation.

Diagnostic reagents that can detect and measure the target biomarkersand sets of biomarkers identified herein and methods for evaluating thelevel or ratios of these target biomarkers vs. their level(s) in avariety of reference standards or controls of different conditions orstages in ovarian cancer are valuable tools in the early detection andmonitoring of ovarian cancer.

I. Definitions

“Patient” or “subject” as used herein means a female mammalian animal,including a human, a veterinary or farm animal, a domestic animal orpet, and animals normally used for clinical research. In one embodiment,the subject of these methods and compositions is a human.

By “biomarker” or “biomarker signature” as used herein is meant a singleprotein or a combination of proteins or peptide fragments thereof, theprotein levels or relative protein levels or ratios of whichsignificantly change (either in an increased or decreased manner) fromthe level or relative levels present in a subject having one physicalcondition or disease or disease stage from that of a reference standardrepresentative of another physical condition or disease stage.Throughout this specification, wherever a particular biomarker isidentified by name, it should be understood that the term “biomarker”includes CLIC4 and/or multiple related proteins of the tropomyosinfamilies. These biomarkers may be combined to form certain sets ofbiomarkers or ligands to biomarkers in diagnostic reagents. Still other“additional” biomarkers are mentioned specifically herein in combinationwith CLIC4 and/or the multiple tropomyosin protein members. Biomarkersdescribed in this specification include any physiological molecularforms, or modified physiological molecular forms, isoforms, pro-forms,and peptide fragments thereof, unless otherwise specified. It isunderstood that all molecular forms useful in this context arephysiological, e.g., naturally occurring in the species. Preferably thepeptide fragments obtained from the biomarkers are unique sequences,such as those exemplified below. However, it is understood thatfragments other than those explicitly identified may be obtained readilyby one of skill in the art in view of the teachings provided herein.

By “isoform” or “multiple molecular form” is meant an alternativeexpression product or variant of a single gene in a given species,including forms generated by alternative splicing, single nucleotidepolymorphisms, alternative promoter usage, alternative translationinitiation small genetic differences between alleles of the same gene,and posttranslational modifications (PTMs) of these sequences.

By “related proteins” or “proteins of the same family” are meantexpression products of different genes or related genes identified asbelonging to a common family. Related proteins in the same biomarkerfamily may or may not share related functions. Related proteins can bereadily identified as having significant sequence identity either overthe entire protein or a significant part of the protein that istypically referred to as a “domain”; typically proteins with at least20% sequence homology or sequence identity can be readily identified asbelonging to the same protein family.

By “homologous protein” is meant an alternative form of a relatedprotein produced from a related gene having a percent sequencesimilarity or identity of greater than 20%, greater than 30%, greaterthan 40%, greater than 50%, greater than 60%, greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, greater than 97%, or greater than 99%.

“Reference standard” as used herein refers to the source of thereference biomarker levels. The “reference standard” is preferablyprovided by using the same assay technique as is used for measurement ofthe subject's biomarker levels in the reference subject or population,to avoid any error in standardization. The reference standard is,alternatively, a numerical value, a predetermined cutpoint, a mean, anaverage, a numerical mean or range of numerical means, a numericalpattern, a ratio, a graphical pattern or a protein abundance profile orprotein level profile derived from the same biomarker or biomarkers in areference subject or reference population. In an embodiment, in whichexpression of nucleic acid sequences encoding the biomarkers is desiredto be evaluated, the reference standard can be an expression level ofone or more biomarkers or an expression profile.

“Reference subject” or “Reference Population” defines the source of thereference standard. In one embodiment, the reference is a human subjector a population of subjects having no ovarian cancer, i.e., healthycontrols or negative controls. In yet another embodiment, the referenceis a human subject or population of subjects with one or more clinicalindicators of ovarian cancer, but who did not develop ovarian cancer. Instill another embodiment, the reference is a human subject or apopulation of subjects having benign ovarian nodules or cysts. In stillanother embodiment, the reference is a human subject or a population ofsubjects who had ovarian cancer, following surgical removal of anovarian tumor. In another embodiment, the reference is a human subjector a population of subjects who had ovarian cancer and were evaluatedfor biomarker levels prior to surgical removal of an ovarian tumor.Similarly, in another embodiment, the reference is a human subject or apopulation of subjects evaluated for biomarker levels followingtherapeutic treatment for ovarian cancer. In still another embodiment,the reference is a human subject or a population of subjects prior totherapeutic treatment for an ovarian cancer. Similarly, in anotherembodiment, the reference human subject or a population of subjectswithout ovarian cancer but which tests positive for a protein level ofCA-125 or HE4. Similarly, in another embodiment, the reference humansubject or a population of subjects with ovarian cancer but which testsnegative for a protein level of CA125 or HE4. In still other embodimentsof methods described herein, the reference is obtained from the sametest subject who provided a temporally earlier biological sample. Thatsample can be pre- or post-therapy or pre- or post-surgery.

Other potential reference standards are obtained from a reference thatis a human subject or a population of subjects having early stageovarian cancer. In another embodiment the reference is a human subjector a population of subjects having advanced stage ovarian cancer. Instill another embodiment, the reference is a human subject or apopulation of subjects having a subtype of epithelial ovarian cancer. Instill another embodiment, the reference is a human subject or apopulation of subjects having serous ovarian cancer or serous papillaryadenocarcinoma. In still another embodiment, the reference is a humansubject or a population of subjects having mucinous ovarian cancer. Instill another embodiment, the reference is a human subject or apopulation of subjects having clear cell ovarian cancer. In stillanother embodiment, the reference is a subject or a population ofsubjects having endometrioid ovarian cancer. In another embodiment, thereference is a human subject or a population of subjects havingMullerian ovarian cancer. In another embodiment, the reference is ahuman subject or a population of subjects having undifferentiatedovarian cancer or an ovarian sarcoma. In another embodiment, thereference standard is a combination of two or more of the abovereference standards.

Selection of the particular class of reference standards, referencepopulation, biomarker levels or profiles depends upon the use to whichthe diagnostic/monitoring methods and compositions are to be put by thephysician and the desired result, e.g., initial diagnosis of ovariancancer or other ovarian condition, clinical management of patients withovarian cancer after initial diagnosis, including, but not limited to,monitoring for reoccurrence of disease or monitoring remission orprogression of the cancer and either before, during or after therapeuticor surgical intervention, selecting among therapeutic protocols forindividual patients, monitoring for development of toxicity or othercomplications of therapy, predicting development of therapeuticresistance, and the like. Such reference standards or controls are thetypes that are commonly used in similar diagnostic assays for otherbiomarkers.

“Sample” as used herein means any biological fluid or tissue thatcontains the ovarian cancer biomarkers identified herein. The mostsuitable samples for use in the methods and with the compositions aresamples which require minimal invasion for testing, e.g., blood samples,including serum, plasma, whole blood, and circulating tumor cells. It isalso anticipated that other biological fluids, such as saliva or urine,vaginal or cervical secretions, and ascites fluids or peritoneal fluidmay be similarly evaluated by the methods described herein. Also,circulating tumor cells or fluids containing them are also suitablesamples for evaluation in certain embodiments of this invention. Thesamples may include biopsy tissue, tumor tissue, surgical tissue,circulating tumor cells, or other tissue. Such samples may further bediluted with saline, buffer or a physiologically acceptable diluent.Alternatively, such samples are concentrated by conventional means. Incertain embodiments, e.g., those in which expression levels of nucleicacid sequences encoding the biomarkers are desired to be evaluated, thesamples may include biopsy tissue, surgical tissue, circulating tumorcells, or other tissue. In one embodiment, the sample is a tumorsecretome, i.e., any fluid or medium containing the proteins secretedfrom the tumor. These shed proteins may be unassociated, associated withother biological molecules, or enclosed in a lipid membrane such as anexosome. In another embodiment, the sample is plasma.

By “significant change in protein level” is meant an increased proteinlevel of a selected biomarker in comparison to that of the selectedreference standard or control or relative to a predetermined cutpoint; adecreased protein level of a selected biomarker in comparison to that ofthe selected reference or control or relative to a predeterminedcutpoint; or a combination of a pattern or relative pattern of certainincreased and/or decreased biomarkers.

The degree of change in biomarker protein level can vary with eachindividual and is subject to variation with each population. Forexample, in one embodiment, a large change, e.g., 2-3 fold increase ordecrease in protein levels of a small number of biomarkers, e.g., from 1to 9 characteristic biomarkers, is statistically significant. In anotherembodiment, a smaller relative change in 10 or more (i.e., about 10, 20,24, 29, or 30 or more biomarkers) is statistically significant. Thedegree of change in any biomarker(s) expression varies with thecondition, such as type of ovarian cancer and with the size or spread ofthe cancer or solid tumor. The degree of change also varies with theimmune response of the individual and is subject to variation with eachindividual. For example, in one embodiment of this invention, a changeat or greater than a 1.2 fold increase or decrease in protein level of abiomarker or more than two such biomarkers, or even 3 or morebiomarkers, is statistically significant. In another embodiment, alarger change, e.g., at or greater than a 1.5 fold, greater than 1.7fold or greater than 2.0 fold increase or a decrease in expression of abiomarker(s) is statistically significant. This is particularly true forcancers without solid tumors. Still alternatively, if a single biomarkerprotein level is significantly increased in biological samples whichnormally do not contain measurable protein levels of the biomarker, suchincrease in a single biomarker level may alone be statisticallysignificant. Conversely, if a single biomarker protein level is normallydecreased or not significantly measurable in certain biological sampleswhich normally do contain measurable protein levels of the biomarker,such decrease in protein level of a single biomarker may alone bestatistically significant.

A change in protein level of a biomarker required for diagnosis ordetection by the methods described herein refers to a biomarker whoseprotein level is increased or decreased in a subject having a conditionor suffering from a disease, specifically ovarian cancer, relative toits expression in a reference subject or reference standard. Biomarkersmay also be increased or decreased in protein level at different stagesof the same disease or condition. The protein levels of specificbiomarkers differ between normal subjects and subjects suffering from adisease, benign ovarian nodules, or cancer, or between various stages ofthe same disease. Protein levels of specific biomarkers differ betweenpre-surgery and post-surgery patients with ovarian cancer. Suchdifferences in biomarker levels include both quantitative, as well asqualitative, differences in the temporal or relative protein level orabundance patterns among, for example, biological samples of normal anddiseased subjects, or among biological samples which have undergonedifferent disease events or disease stages. For the purpose of thisinvention, a significant change in biomarker protein levels whencompared to a reference standard is considered to be present when thereis a statistically significant (p<0.05) difference in biomarker proteinlevel between the subject and reference standard or profile, orsignificantly different relative to a predetermined cut-point.

For example, in one embodiment, the test subject's biomarker(s) levelsare compared with a healthy reference standard. If the subject hasovarian cancer, the selected EOC biomarker(s), e.g., CLIC 4 and/or TPM1,2, 3, and/or 4, will typically show a change in protein level from thelevels in the healthy reference standard, thus permitting diagnosis ofovarian cancer. In another example, these biomarker(s) differentiallychange in protein level (either by increased or decreased protein level)when the biomarker levels or relative levels from the sample of asubject having one of the following conditions is compared to areference subject or population having another of the following physicalconditions. These “conditions” include no ovarian cancer, the presenceof benign ovarian nodules, the presence of an ovarian cancer or subtype,the condition following surgical removal of an ovarian tumor; thecondition prior to surgical removal of an ovarian tumor; the conditionfollowing a specific therapeutic treatment for an ovarian tumor; thecondition prior to a specific therapeutic treatment for an ovariantumor. It is further anticipated that the biomarker(s) expression levelsmay change and the changes may be detected during treatment for ovariancancer. In another embodiment, a condition includes that of a subjecthaving undiagnosed clinical symptoms of abdominal pain or otherabdominal condition of unknown origin. Still other embodiments of“conditions” as defined above include early stage ovarian cancer;advanced stage ovarian cancer, a subtype of epithelial ovarian cancer,serous ovarian cancer; mucinous ovarian cancer, clear cell ovariancancer, endometrioid ovarian cancer, Mullerian ovarian cancer;undifferentiated ovarian cancer, serous papillary adenocarcinoma; andsarcoma.

The term “ligand” refers with regard to protein biomarkers to a moleculethat binds or complexes, with a biomarker protein, molecular form orpeptide, such as an antibody, antibody mimic or equivalent that binds toor complexes with a biomarker identified herein, a molecular form orfragment thereof. In certain embodiments, in which the biomarkerexpression is to be evaluated, the ligand can be a nucleotide sequence,e.g., polynucleotide or oligonucleotide, primer or probe.

As used herein, the term “antibody” refers to an intact immunoglobulinhaving two light and two heavy chains or fragments thereof capable ofbinding to a biomarker protein or a fragment of a biomarker protein.Thus a single isolated antibody or fragment may be a monoclonalantibody, a synthetic antibody, a recombinant antibody, a chimericantibody, a humanized antibody, or a human antibody. The term “antibodyfragment” refers to less than an intact antibody structure, including,without limitation, an isolated single antibody chain, an Fv construct,a Fab construct, an Fc construct, a light chain variable orcomplementarity determining region (CDR) sequence, etc.

As used herein, “labels” or “reporter molecules” are chemical orbiochemical moieties useful for labeling a ligand, e.g., amino acid,peptide sequence, protein, or antibody. “Labels” and “reportermolecules” include fluorescent agents, chemiluminescent agents,chromogenic agents, quenching agents, radionucleotides, enzymes,substrates, cofactors, inhibitors, radioactive isotopes, magneticparticles, and other moieties known in the art. “Labels” or “reportermolecules” are capable of generating a measurable signal and may becovalently or noncovalently joined to a ligand.

As used herein the term “cancer” refers to or describes thephysiological condition in mammals that is typically characterized byunregulated cell growth. More specifically, as used herein, the term“cancer” means any ovarian cancer. In one embodiment, the ovarian canceris an epithelial ovarian cancer or subtype as referred to in“conditions” above. In still an alternative embodiment, the cancer is an“early stage” (I or II) ovarian cancer. In still another embodiment, thecancer is a “late stage” (III or IV) ovarian cancer.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

By “therapeutic reagent” or “regimen” is meant any type of treatmentemployed in the treatment of cancers with or without solid tumors,including, without limitation, chemotherapeutic pharmaceuticals,biological response modifiers, radiation, diet, vitamin therapy, hormonetherapies, gene therapy, surgical resection, etc.

The term “microarray” refers to an ordered arrangement ofbinding/complexing array elements or ligands, e.g. antibodies, on asubstrate.

In the context of the compositions and methods described herein,reference to “at least two,” “at least five,” etc. of the biomarkerslisted in any particular biomarker set means any and all combinations ofthe biomarkers identified. Specific biomarkers for the biomarker profiledo for use in this invention include CLIC4, but may also include anybiomarker, fragment or molecular form, as discussed herein.

By “significant change in expression” is meant an upregulation in theexpression level of a nucleic acid sequence, e.g., genes or transcript,encoding a selected biomarker, in comparison to the selected referencestandard or control; a downregulation in the expression level of anucleic acid sequence, e.g., genes or transcript, encoding a selectedbiomarker, in comparison to the selected reference standard or control;or a combination of a pattern or relative pattern of certain upregulatedand/or down regulated biomarker genes. The degree of change in biomarkerexpression can vary with each individual as stated above for proteinbiomarkers.

The term “polynucleotide,” when used in singular or plural form,generally refers to any polyribonucleotide or polydeoxribonucleotide,which may be unmodified RNA or DNA or modified RNA or DNA. Thus, forinstance, polynucleotides as defined herein include, without limitation,single- and double-stranded DNA, DNA including single- anddouble-stranded regions, single- and double-stranded RNA, and RNAincluding single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or include single- and double-stranded regions. Inaddition, the term “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Theterm “polynucleotide” specifically includes cDNAs. The term includesDNAs (including cDNAs) and RNAs that contain one or more modified bases.In general, the term “polynucleotide” embraces all chemically,enzymatically and/or metabolically modified forms of unmodifiedpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells.

The term “oligonucleotide” refers to a relatively short polynucleotideof less than 20 bases, including, without limitation, single-strandeddeoxyribonucleotides, single- or double-stranded ribonucleotides,RNA:DNA hybrids and double-stranded DNAs. Oligonucleotides, such assingle-stranded DNA probe oligonucleotides, are often synthesized bychemical methods, for example using automated oligonucleotidesynthesizers that are commercially available. However, oligonucleotidescan be made by a variety of other methods, including in vitrorecombinant DNA-mediated techniques and by expression of DNAs in cellsand organisms.

One skilled in the art may readily reproduce the compositions andmethods described herein by use of the amino acid sequences of thebiomarkers and other molecular forms, which are publicly available fromconventional sources.

It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment is also be described using“consisting of” or “consisting essentially of” language. It is to benoted that the term “a” or “an”, refers to one or more, for example, “animmunoglobulin molecule,” is understood to represent one or moreimmunoglobulin molecules. As such, the terms “a” (or “an”), “one ormore,” and “at least one” is used interchangeably herein.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts, which provide one skilled in the art witha general guide to many of the terms used in the present application.

II. Biomarkers and Biomarker Signatures Useful in the Methods andCompositions

The “targets” of the compositions and methods of these inventionsinclude, in one aspect, CLIC4, optionally with other biomarkersidentified herein, fragments, particularly unique fragments thereof, andmolecular forms thereof. In certain embodiments, superior diagnostictests for diagnosing the existence of ovarian cancer utilize at leastone of the ligands that bind or complex with CLIC4, or one of thefragments or molecular forms thereof. In other embodiments, superiordiagnostic tests for distinguishing ovarian cancer from one of theconditions recited above utilize multiple ligands, each individuallydetecting a different specific target biomarker identified herein, orisoform, modified form or peptide thereof. In still other methods, noligand is necessary, e.g., MRM assays.

Thus, in one aspect, the target biomarker of the methods andcompositions described herein is chloride intracellular channel protein4 (CLIC4) or an isoform, pro-form, modified molecular form, or uniquepeptide fragment or nucleic acid fragment thereof. The amino acidsequences for CLIC4 and its molecular forms are publically available,such as in GENBANK. In one embodiment, the biomarker CLIC4 is Uniprot IDno. Z9Y696 or a peptide fragment thereof. Certain fragments of CLIC4that may be useful as targets in the methods and compositions describedherein include one or more peptide fragments, such as, but not limitedto, those identified below. It should be understood that, depending uponthe context, any reference to CLIC4 herein also refers to a peptide andthe molecular form thereof, as well as the nucleotide sequences encodingCLIC4 and/or any of its unique peptides or forms. Among such fragmentsuseful for detection of CLIC4 are

(a) GVVFSVTTVDLK, aa 49-60 of SEQ ID NO: 1 (b)LDEYLNSPLPDEIDENSMEDIK, aa 151-172 of SEQ ID NO: 1 (c)NSRPEANEALER, aa 131-142 of SEQ ID NO: 1 (d)YLTNAYSR, aa 20-27 of SEQ ID NO: 1 (d)EVEIAYSDVAK, aa 139-249 of SEQ ID NO: 1 (e)LFMILWLK, aa 41-48 of SEQ ID NO: 1 (f)DEFTNTCPSDK, aa 228-238 of SEQ ID NO: 1 (g)IEEFLEEVLCPPK, aa 91-103 of SEQ ID NO: 1 (h)FLDGNEMTLADCNLLPK, aa 178-194 of SEQ ID NO: 1 (i)EEDKEPLIELFVK, aa 12-24 of SEQ ID NO: 1 or (j)HPESNTAGMDIFAK, aa 111-124 of SEQ ID NO: 1.

In some embodiments, the methionine in each sequence is the oxidizedform; in other embodiments, the methionine in each sequence is in theunoxidized form.

In another embodiment the target biomarker of the methods andcompositions described herein is Tropomyosin 1 (TPM1), as described inInternational Patent Application No. PCT/US12/54136. The amino acidsequence for TPM1 is publically available, such as in GENBANK. In oneembodiment, an isoform of TPM1 is TPM1, variant 6 (UniProt ID No.Q1ZYL5) or TPM1, variant 8 (UniProt ID No. B7Z596). Certain fragments ofTPM1 may also be useful as targets in the methods and compositionsdescribed herein. It should be understood that, depending upon thecontext, any reference to TPM1 herein also refers to any of its peptidesor molecular forms, as well as the nucleotide sequences encoding TPM1and/or any of the peptides.

In another embodiment the target biomarker of the methods andcompositions described herein is Tropomyosin 2 (TPM2). The amino acidsequence for TPM2 is publically available, such as in GENBANK. Inanother embodiment, an isoform of TPM2 is TPM2 beta chain (UniProt IDNo.UR1H_P07951, UR1H_Q5TCU3, or UR1H_Q5TCU8) or TPM2, isoform 2 (UniProtID No. P07951-2, A7XZE4). Certain fragments of TPM2 may also be usefulas targets in the methods and compositions described herein. It shouldbe understood that, depending upon the context, any reference to TPM2herein also refers to any of its peptides or molecular forms, as well asthe nucleotide sequences encoding TPM2 and/or any of the peptides.

In another embodiment the target biomarker of the methods andcompositions described herein is Tropomyosin 3 (TPM3). The amino acidsequence for TPM3 is publically available, such as in GENBANK. In stillanother embodiment, an isoform of TPM3 is TPM3 alpha-3 chain (UniProt IDNo. P06753, Q5VU59, Q5HYB6, B2RDE1, or E2RB38). Certain fragments ofTPM3 may also be useful as targets in the methods and compositionsdescribed herein. It should be understood that, depending upon thecontext, any reference to TPM3 herein also refers to any of its peptidesor molecular forms, as well as the nucleotide sequences encoding TPM3and/or any of the peptides.

In another embodiment the target biomarker of the methods andcompositions described herein is Tropomyosin 4 (TPM4). The amino acidsequence for TPM4 is publically available, such as in GENBANK. In afurther embodiment, an isoform of TPM4 is TPM4 alpha-4 chain (UniProt IDNo. P6736), or TPM4, Isoform 2 (UniProt ID No. P67936-2). Certainfragments of TPM4 may also be useful as targets in the methods andcompositions described herein, e.g., Table 3. It should be understoodthat, depending upon the context, any reference to TPM4 herein alsorefers to any of its peptides or molecular forms, as well as thenucleotide sequences encoding TPM4 and/or any of the peptides.

In another embodiment the target biomarker of the methods andcompositions described herein is a peptide sequence shared by two ormore members of the related family of tropomyosin proteins TPM1, TPM2,TPM3 and TPM4. A common amino acid sequence for TPM or fragments thereofcan be readily generated using and aligning the publically knownsequences for the native TPM family members. It should be understoodthat, depending upon the context, any reference to common TPM hereinalso refers to any of its fragments or peptides derived therefrom or thecommon sequence generated by use of 2 or more of the TPM relatedproteins to generate the alignment.

In still other embodiments, the target biomarker(s) are multiple membersof the related family of tropomyosin proteins, such as tropomyosin 1(TPM1); tropomyosin 2 (TPM2); tropomyosin 3 (TPM3); tropomyosin 4(TPM4); or a common sequence shared by two or more of these relatedproteins.

In still another embodiment, the target biomarker is actually a set oftargets, which set contains CLIC4 (as defined above) and one or moreadditional EOC target biomarkers. In still another embodiment, thetarget biomarker is actually a set of targets, which set contains two ormore related TPM family proteins or a TPM common or shared peptide orprotein (as defined above) and one or more additional EOC targetbiomarkers. It should be understood that wherever in a following list ofbiomarker sets, a TPM biomarker is identified, it could be replaced orused with a common or shared TPM sequence.

Thus, among other embodiments, a set of biomarkers can be one of thefollowing sets: CLIC4, TPM1 or CLIC4, TPM2 or CLIC4, TPM3 or CLIC4,TPM4, or CLIC4, TPM1, TPM2, or CLIC4, TPM1, TPM2, TPM3, or CLIC4, TPM1,TPM2, TPM3, TPM4, or CLIC4, TPM2, TPM3 or CLIC4, TPM2, TPM3, TPM4, orCLIC4, TPM3, TPM4, or CLIC4, TPM2, TPM4, or CLIC4, TPM1, TPM3.

However, still other additional biomarkers can include any of thebiomarkers identified in International Patent Application No.PCT/US12/54136, such as CLIC 1 or an isoform, pro-form, modifiedmolecular form, or unique peptide fragment or unique nucleic acidfragment thereof. The amino acid sequence for CLIC1 is publicallyavailable, such as in GENBANK. Certain fragments of CLIC1 that may beuseful as targets in the methods and compositions described hereininclude one or more peptide fragments, such as, but not limited to,those identified herein. It should be understood that, depending uponthe context, any reference to CLIC1 herein also refers to any of thesepeptides, or any molecular form of the biomarker, as well as thenucleotide sequences encoding CLIC1 and/or any of the peptides. Amonguseful fragments of CLIC1 include

(a) GVTFNVTTVDTK, aa38-49 of SEQ ID NO: 3; (b)LAALNPESNTAGLDIFAK, aa96-113 of SEQ ID NO: 3; or (c)NSNPALNDNLEK, aa120-131 of SEQ ID NO: 3.

Thus, in another embodiment, a desirable set of target biomarkersincludes, without limitation: CLIC4 and CLIC1, or CLIC4, CLIC1, TPM1, orCLIC4, CLIC1, TPM2, or CLIC4, CLIC1, TPM3, or CLIC4, CLIC1, TPM4, orCLIC4, CLIC1, TPM1, TPM2, or CLIC4, CLIC1, TPM1, TPM2, TPM3, or CLIC4,CLIC1, TPM1, TPM2, TPM3, TPM4 or CLIC4, CLIC1, TPM2, TPM3, or CLIC4,CLIC1, TPM2, TPM3, TPM4, or CLIC4, CLIC1, TPM3, TPM4, or CLIC4, CLIC1,TPM1, TPM3, or CLIC4, CLIC1, TPM2, TPM4. In another embodiment, adesirable set of target biomarkers includes, without limitation: CLIC1,TPM2, or CLIC1, TPM3, or CLIC1, TPM4, or CLIC1, TPM1, TPM2, or CLIC1,TPM1, TPM2, TPM3, or CLIC1, TPM1, TPM2, TPM3, TPM4 or CLIC1, TPM2, TPM3,or CLIC1, TPM2, TPM3, TPM4, or CLIC1, TPM3, TPM4, or CLIC1, TPM1, TPM3,or CLIC1, TPM2, TPM4.

Still other sets of target biomarkers that include either CLIC4 and/ormultiple TPM biomarkers as described above, can further include otherknown EOC biomarkers, which may be added to the above-identified setseither individually or in groups. In certain embodiments, the additionalbiomarker is cathepsin D-30 kDa (CTSD-30) or an isoform, pro-form,modified molecular form, or unique peptide fragment or unique nucleicacid fragment thereof, proteins in the same biomarker family orexpressed from a related gene, having at least 20% sequence homology orsequence identity with CTSD-30. In other embodiments, the additionalbiomarker is peroxieredoxin-6 (PRDX6) or an isoform, pro-form, modifiedmolecular form, or unique peptide fragment or unique nucleic acidfragment thereof, or proteins in the same biomarker family or expressedfrom a related gene, having at least 20% sequence homology or sequenceidentity with PRDX6. In still other embodiments, the additionalbiomarker is the known EOC biomarker CA125, or an isoform, pro-form,modified molecular form, or unique peptide fragment therefrom orproteins in the same biomarker family or expressed from a related gene,having at least 20% sequence homology or sequence identity with CA125.In still other embodiments, the additional biomarker is the known EOCbiomarker HE4, or an isoform, pro-form, modified molecular form, orunique peptide fragment therefrom or proteins in the same biomarkerfamily or expressed from a related gene, having at least 20% sequencehomology or sequence identity with HE4. In other embodiments, theadditional biomarker is bisphosphoglycerate mutase (BPGM) or an isoform,pro-form, modified molecular form, or unique peptide fragment or uniquenucleic acid fragment thereof, or proteins in the same biomarker familyor expressed from a related gene, having at least 20% sequence homologyor sequence identity with BPGM. In other embodiments, the additionalbiomarker is proteasome subunit alpha type-7 (PSMA7) or an isoform,pro-form, modified molecular form, or unique peptide fragment or uniquenucleic acid fragment thereof, or proteins in the same biomarker familyor expressed from a related gene, having at least 20% sequence homologyor sequence identity with PSMA7. In other embodiments, the additionalbiomarker is aldose reductase (AKR1B1) or an isoform, pro-form, modifiedmolecular form, or unique peptide fragment or unique nucleic acidfragment thereof, or proteins in the same biomarker family or expressedfrom a related gene, having at least 20% sequence homology or sequenceidentity with AKR1B1. In other embodiments, the additional biomarker ishomeobox protein (HMX1) or an isoform, pro-form, modified molecularform, or unique peptide fragment or unique nucleic acid fragmentthereof, or proteins in the same biomarker family or expressed from arelated gene, having at least 20% sequence homology or sequence identitywith HMX1. In other embodiments, the additional biomarker is melastatin1 (TRPM1) or an isoform, pro-form, modified molecular form, or uniquepeptide fragment or unique nucleic acid fragment thereof, or proteins inthe same biomarker family or expressed from a related gene, having atleast 20% sequence homology or sequence identity with TRPM1. In otherembodiments, the additional biomarker is protein CutA (CUTA) or anisoform, pro-form, modified molecular form, or unique peptide fragmentor unique nucleic acid fragment thereof, or proteins in the samebiomarker family or expressed from a related gene, having at least 20%sequence homology or sequence identity with CUTA. In other embodiments,the additional biomarker is SERPINB12 protein (SERPINB12), or anisoform, pro-form, modified molecular form, or unique peptide fragmentor unique nucleic acid fragment thereof, or proteins in the samebiomarker family or expressed from a related gene, having at least 20%sequence homology or sequence identity with SERPINB12. In otherembodiments, the additional biomarker is cathepsin D-52 kDa (CTSD-52) oran isoform, pro-form, modified molecular form, or unique peptidefragment or unique nucleic acid fragment thereof, or proteins in thesame biomarker family or expressed from a related gene, having at least20% sequence homology or sequence identity with CTSD-52.

In still other multiple biomarker combinations with either CLIC4 and/ormultiple TPM biomarkers or a TPM common or shared biomarker, include,without limitation, or consist of, the following exemplary combinationsof biomarkers or combinations that include different molecular forms ofthe same biomarker, for diagnosis of ovarian cancer or for monitoringthe progression of the severity of disease or remission of disease:

CLIC4 with one or more of CLIC1, PRDX6, BPGM, PSMA7, AKRIB1, HMX1,TRPM1, CUTA, SERPINB12, CTSD-30 kDa or CTSD-52 kDa; or

CLIC4 and one or multiple TPM 1-4, as above, with one or more of CLIC1,PRDX6, BPGM, PSMA7, AKRIB1, HMX1, TRPM1, CUTA, SERPINB12, CTSD-30 kDa orCTSD-52 kDa; or

CLIC4 with one or more of CLIC1, PRDX6, BPGM, PSMA7, AKRIB1, HMX1,TRPM1, CUTA, SERPINB12, CTSD-30 kDa or CTSD-52 kDa, and CA125 and/orHE4; or

CLIC4 and one or multiple TPM 1-4, as above, with CLIC1, PRDX6, BPGM,PSMA7, AKRIB1, HMX1, TRPM1, CUTA, SERPINB12, CTSD-30 kDa or CTSD-52 kDa,and CA125 and/or HE4.

In still another aspect, a biomarker combination includes, withoutlimitation, or consists of, the following exemplary combinations ofbiomarkers for diagnosis of ovarian cancer or for monitoring theprogression of the severity of disease or remission of disease:

CLIC4, CTSD-30 kDa, and/or CLIC1, and/or PRDX6, or

CLIC4, CTSD-30 kDa, CLIC1, and PRDX6;

CLIC4, CTSD-30 kDa, CLIC1, PRDX6 and at least two TPM proteins selectedfrom TPM1, TPM2, TPM3, TPM4 or common or shared TPM;

CLIC4, CTSD-30 kDa, CLIC1, PRDX6 and BPGM,

CLIC4, CTSD-30 kDa, CLIC1, PRDX6 and PSMA7,

CLIC4, CTSD-30 kDa, CLIC1, PRDX6, BPGM, and at least two TPM proteinsselected from TPM1, TPM2, TPM3, TPM4 or common or shared TPM;

CLIC4, CTSD-30 kDa, CLIC1, PRDX6, PSMA7, and at least two TPM proteinsselected from TPM1, TPM2, TPM3, TPM4 or common or shared TPM;

CLIC4, CTSD-30 kDa, CLIC1, PRDX6, BPGM and PSMA7, and at least two TPMproteins selected from TPM1, TPM2, TPM3, TPM4 or common or shared TPM.

Still other permutations of the biomarkers listed herein may form themultiple biomarker targets or an isoform, pro-form, modified molecularform, or peptide fragment thereof in the compositions and methods ofthis invention. One of skill in the art may readily form appropriatecombinations from the biomarkers listed herein, and from any isoform,pro-form, modified molecular form, posttranslational modification, orunique peptide fragment or unique nucleic acid fragment thereof,proteins in the same biomarker family or expressed from a related gene,having at least 20% sequence homology or sequence identity with aspecifically named biomarker herein. It is further anticipated thatCLIC4 alone, multiple tropomyosins (TPM 1, TPM2, TPM3, TPM4 or common orshared TPM) alone, or CLIC4 in concert with multiple tropomyosins may befurther combined with other known EOC markers to form desirable targetsets.

For example, among desirable biomarker signatures are signatures thatcomprise, or consist of, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25or more of the biomarkers described herein or any other known ovariancancer biomarker or molecular forms or peptides thereof. It iscontemplated that even higher abundance biomarkers (including someidentified in PCT/US12/54136) may be useful when combined in a panel orsignature with the low abundance biomarker CLIC4 and/or multiple TPMs.

As further stated above, the biomarkers/biomarker signatures describedabove, can in another embodiment, refer to nucleic acid sequences, genesand transcripts encoding the biomarkers and expression profiles thereof.

III. Diagnostic Reagents, Devices and Kits

A. Labeled or Immobilized Biomarkers or Peptides or Molecular Forms

Thus, in one aspect, a diagnostic reagent or device comprises thebiomarker chloride intracellular channel protein 4 (CLIC4) or anisoform, pro-form, modified molecular form, or unique peptide fragmentor nucleic acid fragment thereof. In one embodiment, the biomarkerprotein or nucleic acid is associated with a molecule or moiety capablealone or in combination with one or more additional molecules ofgenerating a detectable signal. In another embodiment, the biomarkerprotein or nucleic acid is associated with a substrate and isimmobilized.

In another embodiment, a diagnostic reagent or device comprises multiplemembers of the related family of tropomyosin proteins, such astropomyosin 1 (TPM1); tropomyosin 2 (TPM2); tropomyosin 3 (TPM3);tropomyosin 4 (TPM4); or a common or shared sequence formed of two ormore of these related proteins, or multiple isoforms, pro-forms,modified molecular forms, or unique peptide fragments or nucleic acidfragments thereof. In one embodiment, the biomarker protein or nucleicacid is associated with a molecule or moiety capable alone or incombination with one or more additional molecules of generating adetectable signal. In another embodiment, the biomarker protein ornucleic acid is associated with a substrate and is immobilized.

In another embodiment, a diagnostic reagent or device comprises a CLIC4and one or multiple members of the related family of tropomyosinproteins, or the TPM common or shared sequence, or multiple isoforms,pro-forms, modified molecular forms, or unique peptide fragments ornucleic acid fragments thereof. In one embodiment, the biomarkerproteins or nucleic acid sequences are each associated with a moleculeor moiety capable alone or in combination with one or more additionalmolecules of generating a detectable signal. In another embodiment, thebiomarker protein or nucleic acid is associated with a substrate and isimmobilized. Where the reagent or device contains multiple biomarkersequences, the detectable moieties and signals can each be different, soas to identify different results.

In still other embodiments, the device or reagent designed to identifyCLIC4 and/or multiple TPMs can include additional EOC biomarkerproteins, fragments or nucleic acid sequences, such as the disclosed inInternational Patent Application No. PCT/US12/54136, incorporated byreference herein. Thus, a diagnostic reagent or device as describedherein can comprise 1 to 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20 or morebiomarker sequences.

In still other embodiments, diagnostic reagents or devices for use inthe methods of diagnosing ovarian cancer include CLIC4 and/or multipleTPMs and one or more additional target biomarkers or peptide fragmentsidentified herein, including, but not limited to, isoforms or molecularforms thereof, pro-form, modified molecular form, or unique peptidefragment or nucleic acid fragment thereof, or proteins in the samebiomarker family or expressed from a related gene, having at least 20%sequence homology or sequence identity with the identified biomarker.

In still another embodiment of these sets can include one or moreadditional biomarker that indicates the presence of ovarian cancer in ahuman subject. In certain embodiments, the additional biomarker iscathepsin D-30 kDa (CTSD-30) or an isoform, pro-form, modified molecularform, or unique peptide fragment or unique nucleic acid fragmentthereof, proteins in the same biomarker family or expressed from arelated gene, having at least 20% sequence homology or sequence identitywith CTSD-30. In other embodiments, the additional biomarker isperoxieredoxin-6 (PRDX6) or an isoform, pro-form, modified molecularform, or unique peptide fragment or unique nucleic acid fragmentthereof, or proteins in the same biomarker family or expressed from arelated gene, having at least 20% sequence homology or sequence identitywith PRDX6. In still other embodiments, the additional biomarker isCA125, or an isoform, pro-form, modified molecular form, or uniquepeptide fragment therefrom or proteins in the same biomarker family orexpressed from a related gene, having at least 20% sequence homology orsequence identity with CA125. In other embodiments, the additionalbiomarker is bisphosphoglycerate mutase (BPGM) or an isoform, pro-form,modified molecular form, or unique peptide fragment or unique nucleicacid fragment thereof, or proteins in the same biomarker family orexpressed from a related gene, having at least 20% sequence homology orsequence identity with BPGM. In other embodiments, the additionalbiomarker is proteasome subunit alpha type-7 (PSMA7) or an isoform,pro-form, modified molecular form, or unique peptide fragment or uniquenucleic acid fragment thereof, or proteins in the same biomarker familyor expressed from a related gene, having at least 20% sequence homologyor sequence identity with PSMA7. In other embodiments, the additionalbiomarker is aldose reductase (AKR1B1) or an isoform, pro-form, modifiedmolecular form, or unique peptide fragment or unique nucleic acidfragment thereof, or proteins in the same biomarker family or expressedfrom a related gene, having at least 20% sequence homology or sequenceidentity with AKR1B1. In other embodiments, the additional biomarker ishomeobox protein (HMX1) or an isoform, pro-form, modified molecularform, or unique peptide fragment or unique nucleic acid fragmentthereof, or proteins in the same biomarker family or expressed from arelated gene, having at least 20% sequence homology or sequence identitywith HMX1. In other embodiments, the additional biomarker is melastatin1 (TRPM1) or an isoform, pro-form, modified molecular form, or uniquepeptide fragment or unique nucleic acid fragment thereof, or proteins inthe same biomarker family or expressed from a related gene, having atleast 20% sequence homology or sequence identity with TRPM1. In otherembodiments, the additional biomarker is protein CutA (CUTA) or anisoform, pro-form, modified molecular form, or unique peptide fragmentor unique nucleic acid fragment thereof, or proteins in the samebiomarker family or expressed from a related gene, having at least 20%sequence homology or sequence identity with CUTA. In other embodiments,the additional biomarker is SERPINB12 protein (SERPINB12), or anisoform, pro-form, modified molecular form, or unique peptide fragmentor unique nucleic acid fragment thereof, or proteins in the samebiomarker family or expressed from a related gene, having at least 20%sequence homology or sequence identity with SERPINB12. In otherembodiments, the additional biomarker is cathepsin D-52 kDa (CTSD-52) oran isoform, pro-form, modified molecular form, or unique peptidefragment or unique nucleic acid fragment thereof, or proteins in thesame biomarker family or expressed from a related gene, having at least20% sequence homology or sequence identity with CTSD-52.

In such embodiments of the diagnostic reagents and devices, at least oneof the biomarkers is associated with a detectable label or portion of adetectable label system. In another embodiment, a diagnostic reagentincludes one or more target biomarker or peptide fragment identifiedherein, immobilized on a substrate. In still another embodiment,combinations of such labeled or immobilized biomarkers are suitablereagents and components of a diagnostic kit or device. In anotheraspect, suitable embodiments of such labeled or immobilized reagentsinclude at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 20 or morebiomarkers identified herein or their unique peptide fragments.

In one aspect the reagent, device or kit as described above furthercomprises or consists of ligands that individually specifically complexwith, bind to, or quantitatively detect or identify multiple isoforms ormultiple related proteins of any of biomarkers mentioned herein. Inanother aspect, the reagent, device or kit comprises or consists ofligands that individually specifically complex with, bind to, orquantitatively detect or identify two or more biomarkers identified inthe groups specifically discussed above. Still other diagnostic reagentsare the surrogate peptides used for the MRM assays, such as, but notlimited to, the peptides disclosed herein.

Any combination of labeled or immobilized biomarkers as described abovecan be assembled in a diagnostic kit or device for the purposes ofdiagnosing ovarian cancer, such as those combinations of biomarkersdiscussed herein.

For these reagents, the labels may be selected from among many knowndiagnostic labels, including those described above. Similarly, thesubstrates for immobilization in a device may be any of the commonsubstrates, glass, plastic, a microarray, a microfluidics card, a chip,a bead or a chamber.

B. Labeled or Immobilized Ligands that Bind or Complex with theBiomarkers

In another aspect, a diagnostic reagent or device comprises a ligandcapable of specifically complexing with, binding to, or quantitativelydetecting or identifying the biomarker chloride intracellular channelprotein 4 (CLIC4) or an isoform, pro-form, modified molecular form, orunique peptide fragment or nucleic acid fragment thereof. In oneembodiment, the ligand is associated with a molecule or moiety capablealone or in combination with one or more additional molecules ofgenerating a detectable signal. In another embodiment, the ligand isassociated with a substrate on which the ligand is immobilized.

In still other embodiments, the diagnostic reagent or device comprises aset of multiple ligands, each ligand individually capable ofspecifically complexing with, binding to, or quantitatively detecting oridentifying a single biomarker or an isoform, pro-form, modifiedmolecular form, or unique peptide fragment or nucleic acid fragmentthereof. In another embodiment, a diagnostic reagent or device comprisesa ligand capable of specifically complexing with, binding to, orquantitatively detecting or identifying multiple members of the relatedfamily of tropomyosin proteins, such as tropomyosin 1 (TPM1);tropomyosin 2 (TPM2); tropomyosin 3 (TPM3); tropomyosin 4 (TPM4); or acommon or shared sequence within two or more of these related proteins.In another embodiment, a diagnostic reagent or device comprises multipleligands, each ligand capable of identifying a different TPM familymember.

In another embodiment, a diagnostic reagent or device comprises a ligandcapable of specifically complexing with, binding to, or quantitativelydetecting or identifying CLIC4 and one or more ligands capable ofspecifically complexing with, binding to, or quantitatively detecting oridentifying individually one or multiple members of the related familyof tropomyosin proteins, or the TPM common or shared sequence. In stillother embodiments, the device or reagent designed to identify CLIC4and/or multiple TPMs can contain ligand capable of specificallycomplexing with, binding to, or quantitatively detecting or identifyingother additional EOC biomarkers, such as the unique peptides directed tothe above biomarkers and as disclosed in International PatentApplication No. PCT/US12/54136, incorporated by reference herein.

Thus, a diagnostic reagent or device as described herein can comprise 1to 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20 or more such ligands to identify anequivalent number of biomarkers. In still other embodiments, diagnosticreagents or devices for use in the methods of diagnosing ovarian cancerinclude ligand capable of specifically complexing with, binding to, orquantitatively detecting or identifying CLIC4 and/or multiple TPMs andone or more additional target biomarkers or peptide fragments identifiedherein, including, but not limited to, isoforms or molecular formsthereof, pro-form, modified molecular form, or unique peptide fragmentor nucleic acid fragment thereof, or proteins in the same biomarkerfamily or expressed from a related gene, having at least 20% sequencehomology or sequence identity with the identified biomarker.

In still another embodiment, one or more of said ligands in thediagnostic reagent or device is associated with a molecule or moietycapable alone or in combination with one or more additional molecules ofgenerating a detectable signal, a detectable label or portion of adetectable label system. In another embodiment, a diagnostic reagentincludes one or more ligand capable of specifically complexing with,binding to, or quantitatively detecting or identifying target biomarkeror peptide fragment identified herein, immobilized on a substrate. Instill another embodiment, combinations of such labeled or immobilizedligands are suitable reagents and components of a diagnostic kit ordevice.

In one aspect the reagent, device or kit comprises or consists ofligands that complex with, bind to, or quantitatively detect or identifymultiple isoforms or multiple related proteins of any of biomarkersmentioned herein. In another aspect, the reagent, device or kitcomprises or consists of ligands that individually specifically complexwith, bind to, or quantitatively detect or identify two or morebiomarkers in a biomarker family. Still other diagnostic reagents arethe ligands to the surrogate peptides used for the MRM assays, such as,but not limited to, the peptides disclosed herein.

Any combination of labeled or immobilized ligands can be assembled in adiagnostic kit or device for the purposes of diagnosing ovarian cancer,such as those combinations of ligands to target biomarker sets discussedherein.

For these reagents, the labels may be selected from among many knowndiagnostic labels, including those described above. Similarly, thesubstrates for immobilization in a device may be any of the commonsubstrates, glass, plastic, a microarray, a microfluidics card, a chip,a bead or a chamber.

In another embodiment, the diagnostic reagent or device includes aligand that binds to or complexes with a biomarker as identified aboveor a unique peptide thereof, or a molecular form thereof, or a relatedprotein family member expressed by a different, related gene, or acombination of such ligands. In certain aspects, the diagnostic reagentor device includes ligands which can include an antibody or fragment ofan antibody, an antibody mimic, a synthetic antibody, a single chainantibody or an equivalent that binds to or complexes with a singlebiomarker, said ligand optionally associated with a detectable label orwith a substrate. Such antibodies may be presently extant in the art orpresently used commercially, such as those available as part ofcommercial antibody sandwich ELISA assay kits or that may be developedby techniques now common in the field of immunology. A recombinantmolecule bearing the binding portion of a biomarker antibody, e.g.,carrying one or more variable chain CDR sequences that bind e.g., CLIC4,TPM1, TPM2, TPM3, TPM4, PRDX6, CTSD-30, CDSD-52, CLIC1, etc. may also beused in a diagnostic assay. As used herein, the term “antibody” may alsorefer, where appropriate, to a mixture of different antibodies orantibody fragments that bind to the selected biomarker. Such differentantibodies may bind to different biomarkers or different portions of thesame biomarker protein than the other antibodies in the mixture. Suchdifferences in antibodies used in the assay may be reflected in the CDRsequences of the variable regions of the antibodies. Such differencesmay also be generated by the antibody backbone, for example, if theantibody itself is a non-human antibody containing a human CDR sequence,or a chimeric antibody or some other recombinant antibody fragmentcontaining sequences from a non-human source. Antibodies or fragmentsuseful in the compositions or methods described herein may be generatedsynthetically or recombinantly, using conventional techniques or may beisolated and purified from plasma or further manipulated to increase thebinding affinity thereof. It should be understood that any antibody,antibody fragment, or mixture thereof that binds one of the biomarkersdescribed herein or a particular sequence of the selected biomarker orpeptide fragment thereof may be employed in the compositions andmethods, regardless of how the antibody or mixture of antibodies wasgenerated. Various forms of antibody, e.g., polyclonal, monoclonal,recombinant, chimeric, as well as fragments and components (e.g., CDRs,single chain variable regions, etc.) or antibody mimics or equivalentsmay be used in place of antibodies. The ligand itself may be labeled orimmobilized.

In another embodiment, the reagent ligands are nucleotide sequences, thediagnostic reagent is a polynucleotide or oligonucleotide sequence thathybridizes to gene, gene fragment, gene transcript or nucleotidesequence encoding a biomarker discussed herein or encoding a uniquepeptide thereof. Such a polynucleotide/oligonucleotide can be a probe orprimer, and may itself be labeled or immobilized. In one embodiment,ligand-hybridizing polynucleotide or oligonucleotide reagent(s) are partof a primer-probe set, and the kit comprises both primer and probe. Eachsaid primer-probe set amplifies a different gene, gene fragment or geneexpression product that encodes a different biomarker discussed herein,optionally including one or more additional known biomarkers, also asdescribed above. For use in the compositions the PCR primers and probesare preferably designed based upon intron sequences present in thebiomarker gene(s) to be amplified selected from the gene expressionprofile. The design of the primer and probe sequences is within theskill of the art once the particular gene target is selected. Theparticular methods selected for the primer and probe design and theparticular primer and probe sequences are not limiting features of thesecompositions. A ready explanation of primer and probe design techniquesavailable to those of skill in the art is summarized in U.S. Pat. No.7,081,340, with reference to publically available tools such as DNABLAST software, the Repeat Masker program (Baylor College of Medicine),Primer Express (Applied Biosystems); MGB assay-by-design (AppliedBiosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3on the WWW for general users and for biologist programmers and otherpublications.

In general, optimal PCR primers and probes used in the compositionsdescribed herein are generally 17-30 bases in length, and contain about20-80%, such as, for example, about 50-60% G+C bases. Meltingtemperatures of between 50 and 80° C., e.g. about 50 to 70° C. aretypically preferred.

In one embodiment, such a ligand binding to a protein biomarker or aunique peptide contained therein, can be an antibody which specificallybinds a single biomarker such as CLIC4, or a unique peptide in thatsingle biomarker or a nucleic acid sequence which hybridizes to thenucleic acid sequence encoding CLIC4 or its unique peptide. In oneembodiment, such a ligand desirably binds to a protein biomarker or aunique peptide contained therein, and can be an antibody whichspecifically binds to one or more of the TPM family members, e.g., TPM1,2, 3, or 4 individually, collectively, or individually to a uniquepeptide in that single TPM biomarker, or to a TPM common or sharedbiomarker. In another embodiment, such a ligand can hybridize to thenucleic acid sequence encoding the protein biomarker or a unique peptidecontained therein, where the biomarker is one or more of the TPM familymembers, e.g., TPM1, 2, 3, or 4 individually, collectively, orindividually to a nucleic acid encoding a unique peptide in that singleTPM biomarker, or to a TPM common or shared biomarker.

In another embodiment, suitable labeled or immobilized reagents includeat least 2, 3, 4, 5, 6, 7 8, 9, 10 or 11 or more ligands or antibodies,in which each ligand binds to or complexes with a single biomarkerprotein/peptide, fragment, or molecular form of the biomarker(s) listedin detail above. In some of the embodiments in which the combination ofbiomarkers including those listed specifically above or another knownadditional biomarker that may be in higher abundance in serum, ligandsto each of these additional biomarkers may be employed in the diagnosticreagent.

Any combination of labeled or immobilized biomarker ligands can beassembled in a diagnostic kit or device for the purposes of diagnosingovarian cancer.

Thus, a kit or device can contain multiple reagents or one or moreindividual reagents. For example, one embodiment of a compositionincludes a substrate upon which the biomarkers or ligands areimmobilized. In another embodiment, the kit also contains optionaldetectable labels, immobilization substrates, optional substrates forenzymatic labels, as well as other laboratory items.

The diagnostic reagents, devices, or kits compositions based on thebiomarkers or fragments described herein or including the ligandsthereto, optionally associated with detectable labels, can be presentedin the format of a microfluidics card, a chip or chamber, a bead or akit adapted for use with assays formats such as sandwich ELISAs,multiple protein assays, platform multiplex ELISAs, such as the BioRadLuminex platform, mass spectrometry quantitative assays, or PCR, RT-PCRor Q PCR techniques.

The selection of the ligands, biomarker sequences, their length,suitable labels and substrates used in the reagents and kits are routinedeterminations made by one of skill in the art in view of the teachingsherein of which biomarkers form signatures suitable for the diagnosis ofovarian cancer. Assembly of the ligands and biomarkers discussed herein,attachment to a substrate, and assembly into the form of a microarray, amicrofluidics card, a chip, a bead, or a chamber employ techniques knownin the art.

IV. Methods for Diagnosing or Monitoring Ovarian Cancer

In another embodiment, a method for diagnosing or detecting ormonitoring the progress of ovarian cancer and treatment of ovariancancer in a subject comprises, or consists of, a variety of steps.

A. Sample Preparation

The test sample is obtained from a human subject who is to undergo thetreatment or is in the process of being treated. The subject's samplecan in one embodiment be provided before initial diagnosis, so that themethod is performed to diagnose the existence of an ovarian cancer. Inanother embodiment, depending upon the reference standard and markersused, the method is performed to diagnosis the stage of ovarian cancer.In another embodiment, depending upon the reference standard and markersused, the method is performed to diagnosis the type or subtype ofovarian cancer from the types and subtypes identified above. In anotherembodiment, the subject's sample can be provided after a diagnosis, sothat the method is performed to monitor progression of an ovariancancer. In another embodiment, the sample can be provided prior tosurgical removal of an ovarian tumor or prior to therapeutic treatmentof a diagnosed ovarian cancer and the method used to thereafter monitorthe effect of the treatment or surgery, and to check for relapse. Inanother embodiment, the sample can be provided following surgicalremoval of an ovarian tumor or following therapeutic treatment of adiagnosed ovarian cancer, and the method performed to ascertain efficacyof treatment or relapse. In yet another embodiment the sample may beobtained from the subject periodically during therapeutic treatment foran ovarian cancer, and the method employed to track efficacy of therapyor relapse. In yet another embodiment the sample may be obtained fromthe subject periodically during therapeutic treatment to enable thephysician to change therapies or adjust dosages. In one or more of theseembodiments, the subject's own prior sample can be employed in themethod as the reference standard.

Preferably where the sample is a fluid, e.g., blood, serum or plasma,obtaining the sample involves simply withdrawing and preparing thesample in traditional fashion for contact with the diagnostic reagent.Where the sample is a tissue or tumor sample, it may be prepared inconventional manner for contact with the diagnostic reagent.

The method further involves contacting the sample obtained from a testsubject with a diagnostic reagent as described above under conditionsthat permit the reagent to bind to or complex with one or morebiomarker(s), e.g., CLIC4 and/or multiple TPM biomarkers, and/oradditional biomarkers which may be present in the sample. This methodmay employ any of the suitable diagnostic reagents or kits orcompositions described above.

B. Measuring Biomarker Levels

Thereafter, a suitable assay is employed to detect or measure in thesample the protein level (actual or relative) of one or morebiomarker(s), including CLIC4 and/or multiple TPMs, and/or additionalbiomarkers discussed herein. Alternatively, a suitable assay is employedto generate a protein abundance profile (actual or relative or ratiosthereof) of multiple biomarkers discussed herein from the sample or ofmultiple different molecular forms of the same biomarker or both. Inanother embodiment, the above method further includes measuring in thebiological sample of the subject the protein level of one or moreadditional biomarkers, such as CTSD-30 kDa, CA125 or other known ovariancancer biomarker identified herein or other known EOC biomarker known inthe art.

In another embodiment, the above method further includes measuring inthe biological sample of the subject the protein levels of two or moreadditional biomarkers which form with CLIC4 and/or multiple or common orshared TPMs, a biomarker protein abundance signature for ovarian cancer.In one embodiment, the measurement of all target biomarkers occurs in asingle sample. In another embodiment, the measurement of all targetbiomarkers occurs in a multiple samples from a single patient. It shouldbe understood that the measurement of all biomarkers need not occursimultaneously or in the same assay. Results from multiple assays may becombined providing that they are performed within a reasonable time forcomparison with the other target biomarker levels.

The measurement of the biomarker(s) in the biological sample may employany suitable ligand, e.g., antibody, antibody mimic or equivalent (orantibody to any second biomarker) to detect the biomarker protein, asdescribed above. Similarly, the antibodies may be tagged or labeled withreagents capable of providing a detectable signal, depending upon theassay format employed. Such labels are capable, alone or in concert withother compositions or compounds, of providing a detectable signal. Wheremore than one antibody is employed in a diagnostic method for a singlebiomarker, e.g., such as in a sandwich ELISA, the labels are desirablyinteractive to produce a detectable signal. Most desirably, the label isdetectable visually, e.g. colorimetrically. A variety of enzyme systemsoperate to reveal a colorimetric signal in an assay, e.g., glucoseoxidase (which uses glucose as a substrate) releases peroxide as aproduct that in the presence of peroxidase and a hydrogen donor such astetramethyl benzidine (TMB) produces an oxidized TMB that is seen as ablue color. Other examples include horseradish peroxidase (HRP) oralkaline phosphatase (AP), and hexokinase in conjunction withglucose-6-phosphate dehydrogenase that reacts with ATP, glucose, andNAD+ to yield, among other products, NADH that is detected as increasedabsorbance at 340 nm wavelength.

Other label systems that may be utilized in the methods and devices ofthis invention are detectable by other means, e.g., colored latexmicroparticles (Bangs Laboratories, Indiana) in which a dye is embeddedmay be used in place of enzymes to provide a visual signal indicative ofthe presence of the resulting selected biomarker-antibody complex inapplicable assays. Still other labels include fluorescent compounds,radioactive compounds or elements. Preferably, an anti-biomarkerantibody is associated with, or conjugated to a fluorescent detectablefluorochromes, e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE), allophycocyanin (APC), coriphosphine-O (CPO) or tandem dyes,PE-cyanin-5 (PC5), and PE-Texas Red (ECD). Commonly used fluorochromesinclude fluorescein isothiocyanate (FITC), phycoerythrin (PE),allophycocyanin (APC), and also include the tandem dyes, PE-cyanin-5(PC5), PE-cyanin-7 (PC7), PE-cyanin-5.5, PE-Texas Red (ECD), rhodamine,PerCP, fluorescein isothiocyanate (FITC) and Alexa dyes. Combinations ofsuch labels, such as Texas Red and rhodamine, FITC+PE, FITC+PECy5 andPE+PECy7, among others may be used depending upon assay method.

Detectable labels for attachment to antibodies useful in diagnosticassays and devices of this invention may be easily selected from amongnumerous compositions known and readily available to one skilled in theart of diagnostic assays. The biomarker-antibodies or fragments usefulin this invention are not limited by the particular detectable label orlabel system employed. Thus, selection and/or generation of suitablebiomarker antibodies with optional labels for use in this invention iswithin the skill of the art, provided with this specification, thedocuments incorporated herein, and the conventional teachings ofimmunology.

Similarly the particular assay format used to measure the selectedbiomarker in a biological sample may be selected from among a wide rangeof protein assays, such as described in the examples below. Suitableassays include enzyme-linked immunoassays, sandwich immunoassays,homogeneous assays, immunohistochemistry formats, or other conventionalassay formats. In one embodiment, a serum/plasma sandwich ELISA isemployed in the method. In another embodiment, a mass spectrometry-basedassay is employed. In another embodiment, a MRM assay is employed, inwhich antibodies are used to enrich the biomarker in a manner analogousto the capture antibody in sandwich ELISAs. One of skill in the art mayreadily select from any number of conventional immunoassay formats toperform this invention.

Other reagents for the detection of protein in biological samples, suchas peptide mimetics, synthetic chemical compounds capable of detectingthe selected biomarker may be used in other assay formats for thequantitative detection of biomarker protein in biological samples, suchas high pressure liquid chromatography (HPLC), immunohistochemistry,etc.

Employing ligand binding to the biomarker proteins or multiplebiomarkers forming the signature enables more precise quantitativeassays, as illustrated by the multiple reaction monitoring (MRM) massspectrometry (MS) assays. As an alternative to specific peptide-basedMRM-MS assays that can distinguish specific protein isoforms andproteolytic fragments, the knowledge of specific molecular forms ofbiomarkers allows more accurate antibody-based assays, such as sandwichELISA assays or their equivalent. Frequently, the isoform specificity,protein domain specificity and affects of posttranslationalmodifications on binding of immune reagents used in pre-clinical (andsome clinical) diagnostic tests are not well defined. MRM-MS assays wereused to quantitative the levels of a number of the low abundancebiomarkers in samples, as discussed in the examples.

In one embodiment, suitable assays for use in these methods includeimmunoassays using antibodies or ligands to the above-identifiedbiomarkers and biomarker signatures. In another embodiment, a suitableassay includes a multiplexed MRM based assay for two more biomarkersthat include one or more of the proteins/unique peptides describedherein. It is anticipated that ultimately the platform most likely to beused in clinical assays will be multi-plexed or parallel sandwich ELISAassays or their equivalent, primarily because this platform is thetechnology most commonly used to quantify blood proteins in clinicallaboratories. MRM MS assays may continue to be used productively to helpevaluate the isoform/molecular form specificity of any existingimmunoassays or those developed in the future. In addition, multiplexedquantitative MS assays such as MRM MS may replace ELISA assays inclinical laboratories in some situations.

C. Detection of a Change in Biomarker Abundance Level and Diagnosis

The protein level of the one or more biomarker(s) in the subject'ssample or the protein abundance profile of multiple said biomarkers asdetected by the use of the assays described above is then compared withthe level of the same biomarker or biomarkers in a reference standard orreference profile. In one embodiment, the comparing step of the methodis performed by a computer processor or computer-programmed instrumentthat generates numerical or graphical data useful in the appropriatediagnosis of the condition. Optionally, the comparison may be performedmanually.

The detection or observation of a change in the protein level of abiomarker or biomarkers in the subject's sample from the same biomarkeror biomarkers in the reference standard can indicate an appropriatediagnosis. An appropriate diagnosis can be identifying a risk ofdeveloping ovarian cancer, a diagnosis of ovarian cancer (or stage ortype thereof), a diagnosis or detection of the status of progression orremission of ovarian cancer in the subject following therapy or surgery,a determination of the need for a change in therapy or dosage oftherapeutic agent. The method is thus useful for early diagnosis ofdisease, for monitoring response or relapse after initial diagnosis andtreatment or to predict clinical outcome or determine the best clinicaltreatment for the subject.

In one embodiment, the change in protein level of each biomarker caninvolve an increase of a biomarker or multiple biomarkers in comparisonto the specific reference standard. In one embodiment, the biomarkerCLIC4 is increased in a subject sample from a patient having ovariancancer when compared to the levels of these biomarkers from a healthyreference standard. In another embodiment, the biomarkers are increasedin a subject sample from a patient having ovarian cancer prior totherapy or surgery, when compared to the levels of these biomarkers froma post-surgery or post-therapy reference standard.

In another embodiment, the change in protein level of each biomarker caninvolve a decrease of a biomarker or multiple biomarkers in comparisonto the specific reference standard. In one embodiment, the biomarkersare decreased in a subject sample from a patient having ovarian cancerfollowing surgical removal of a tumor or followingchemotherapy/radiation when compared to the levels of these biomarkersfrom a pre-surgery/pre-therapy ovarian cancer reference standard or areference standard which is a sample obtained from the same subjectpre-surgery or pre-therapy.

In still other embodiments, the changes in protein levels of thebiomarkers may be altered in characteristic ways if the referencestandard is a particular type of ovarian cancer, e.g., serous,epithelial, mucinous or clear cell, or if the reference standard isderived from benign ovarian cysts or nodules.

The results of the methods and use of the compositions described hereinmay be used in conjunction with clinical risk factors to help physiciansmake more accurate decisions about how to manage patients with ovariancancers. Another advantage of these methods and compositions is thatdiagnosis may occur earlier than with more invasive diagnostic measures.

D. Alternative Assay Embodiments

In an alternative embodiment, the method of diagnosis or risk ofdiagnosis involves using the nucleic acid hybridizing reagent ligandsdescribed above to detect a significant change in expression level ofthe subject's sample biomarker or biomarkers from that in a referencestandard or reference expression profile which indicates a diagnosis,risk, or the status of progression or remission of ovarian cancer in thesubject. These methods may be performed in other biological samples,e.g., biopsy tissue samples, tissue removed by surgery, or tumor cellsamples, including circulating tumor cells isolated from the blood, todetect or analyze a risk of developing an ovarian cancer, as well as adiagnosis of same. Such methods are also known in the art and includecontacting a sample obtained from a test subject with a diagnosticreagent comprising a ligand which is a nucleotide sequence capable ofhybridizing to a nucleic acid sequence encoding a biomarker or biomarkercombination described herein, e.g., CLIC4 and/or multiple TPM proteins,said ligand associated with a detectable label or with a substrate.Thereafter one would detect or measure in the sample or from anexpression profile generated from the sample, the expression levels ofone or more of the biomarkers or ratios thereof. The expression level(s)of the biomarker(s) in the subject's sample or from an expressionprofile or ratio of multiple said biomarkers are then compared with theexpression level of the same biomarker or biomarkers in a referencestandard. A significant change in expression level of the subject'ssample biomarker or biomarkers from that in the reference standardindicates a diagnosis, risk, or the status of progression or remissionof ovarian cancer in the subject.

Suitable assay methods include methods based on hybridization analysisof polynucleotides, methods based on sequencing of polynucleotides,proteomics-based methods or immunochemistry techniques. The mostcommonly used methods known in the art for the quantification of mRNAexpression in a sample include northern blotting and in situhybridization; RNAse protection assays; and PCR-based methods, such asreverse transcription polymerase chain reaction (RT-PCR) or qPCR.Alternatively, antibodies may be employed that can recognize specificDNA-protein duplexes. The methods described herein are not limited bythe particular techniques selected to perform them. Exemplary commercialproducts for generation of reagents or performance of assays includeTRI-REAGENT, Qiagen RNeasy mini-columns, MASTERPURE Complete DNA and RNAPurification Kit (EPICENTRE®, Madison, Wis.), Paraffin Block RNAIsolation Kit (Ambion, Inc.) and RNA Stat-60 (Tel-Test), theMassARRAY-based method (Sequenom, Inc., San Diego, Calif.), differentialdisplay, amplified fragment length polymorphism (iAFLP), and BeadArray™technology (Illumina, San Diego, Calif.) using the commerciallyavailable Luminex100 LabMAP system and multiple color-coded microspheres(Luminex Corp., Austin, Tex.) and high coverage expression profiling(HiCEP) analysis.

The comparison of the quantitative or relative expression levels of thebiomarkers may be done analogously to that described above for thecomparison of protein levels of biomarkers.

E. Non-Ligand-Based Analysis

In another aspect, a method for diagnosing or detecting or monitoringthe progress of ovarian cancer in a subject involves non-ligand basedmethods, such as mass spectrometry. For example, proteins in abiological sample obtained from a test subject may be contacted with achemical or enzymatic agent and the proteins, including the biomarkerscontained therein fragmented in the sample. The digested sample orportions thereof are injected into a mass spectrometer and the proteinlevels or ratios of one or more of the biomarker or biomarkercombinations described herein, optionally with other known biomarkers,modified molecular forms, peptides and unique peptides or ratiosthereof, are quantitatively identified or measured by mass spectrometry.The protein levels of the biomarkers in the subject's sample are thencompared with the level of the same biomarker or biomarkers in areference standard or to a predetermined cutoff derived from thereference standard. In one embodiment, the agent is a proteolyticenzyme. In another embodiment, the agent is trypsin.

A significant change in protein level of the subject's sample biomarkeror biomarkers from that in the reference standard or from apredetermined cutoff indicates a diagnosis, risk, or the status ofprogression or remission of ovarian cancer in the subject.

Thus, the various methods, devices and steps described above can beutilized in an initial diagnosis of ovarian cancer or other ovariancondition, as well as in clinical management of patients with ovariancancer after initial diagnosis. Uses in clinical management of thevarious devices, reagents and assay methods, include without limitation,monitoring for reoccurrence of disease or monitoring remission orprogression of the cancer and either before, during or after therapeuticor surgical intervention, selecting among therapeutic protocols forindividual patients, monitoring for development of toxicity or othercomplications of therapy, and predicting development of therapeuticresistance.

In one embodiment, the method involves enriching the biomarker proteinor one or more peptides produced by specific proteolysis in the sampleby contacting the sample with an antibody prior to injecting into a massspectrometer in a manner analogous to a capture antibody in aconventional sandwich ELISA. In another embodiment, the method involvesdepleting the sample of non-target proteins prior to injecting sampleinto a mass spectrometer. The depletion may also be performed usingantibodies to the non-targets. The method described herein may useliquid chromatographic mass spectrometry, such as HPLC. One such methodis described in detail in the Examples below.

F. Illustrative Embodiments

In one embodiment, a method for diagnosing or detecting or monitoringthe progress of ovarian cancer in a subject comprises contacting asample obtained from a test subject with a diagnostic reagent or devicecomprising a ligand capable of specifically complexing with, binding to,or quantitatively detecting or identifying the biomarker chlorideintracellular channel protein 4 (CLIC4) or an isoform, pro-form,modified molecular form, or unique peptide fragment or nucleic acidfragment thereof. Thereafter, the method involves detecting or measuringin the sample or from a protein level profile generated from the sample,the protein levels of the CLIC4 biomarker. Another step includescomparing the protein levels of the CLIC4 biomarker in the subject'swith the level of the same biomarker in a reference standard. Asignificant change in protein level of the subject's sample from that inthe reference standard indicates a diagnosis, risk, or the status ofprogression or remission of ovarian cancer in the subject.

In another aspect, the method comprises detecting or measuring in thesample or from a protein level profile generated from the sample, theprotein levels of one or more additional ovarian cancer biomarkers; andcomparing the protein levels of the CLIC4 biomarker in relation to thelevels of the additional biomarkers in the subject's sample with thesame biomarkers in a reference standard or profile.

In another aspect, the method uses as a reference standard a mean, anaverage, a numerical mean or range of numerical means, a numericalpattern, a ratio, a graphical pattern or a protein level profile derivedfrom the same biomarker or biomarkers in a reference subject orreference population. In certain embodiments, the change in proteinlevel of each biomarker comprises an increase in comparison to saidreference or control or a decrease in comparison to said reference orcontrol.

Use of such methods permits early diagnosis of disease, monitoringrelapse after initial diagnosis and treatment, predicting clinicaloutcome, or determining the best clinical treatment.

IV. Examples

As discussed in detail in the examples below, it was discovered that asingle member of the chloride intracellular channel proteins, CLIC4,which is expressed from a related but different gene than CLIC1, wasreadily and with great sensitivity, detectable in ovarian cancer patientsera. In additional aspects, related tropomyosin proteins expressed froma gene other than the gene expressing TPM1 and its isoforms, were alsodetectable in ovarian cancer patent sera. A multiplexed, label-freemultiple reaction monitoring (MRM) assay was established to targetpeptides specific to the detected CLIC and TPM biomarkers, and werequantitated for ovarian cancer patients, patients with benign disease,and normal donors. In addition to CLIC1 and TPM1, which were biomarkerproteins discovered in a xenograft mouse model (see PCT/US12/54136,cited above), CLIC4, TPM2, TPM3, and TPM4 were present in ovarian cancerpatient sera at significantly elevated levels compared with controls.These additional biomarkers may be superior to the previously identifiedbiomarkers at discriminating between cancer and noncancer patients.

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations that become evident as a result of the teaching providedherein.

Example 1 Materials and Methods

A. Human Serum Collection and Processing

Sera from patients with benign ovarian tumors and from late-stageovarian cancer patients were collected at the University of Turin,Turin, Italy, at the time of diagnosis and have been previouslydescribed (Tang et al, 2012, cited above). Control serum samples werecollected from healthy, post-menopausal female donors at The WistarInstitute, Philadelphia, Pa. All specimens were processed in compliancewith Institutional Review Board (IRB) and Health Insurance Portabilityand Accountability Act (HIPAA) requirements. Control and patient serumsamples were processed for MS analysis either individually or as pools,as previously described.²¹ Dithiothreitol (DTT) was obtained from GEHealthcare (Piscataway, N.J.); and iodoacetamide from Sigma-Aldrich (St.Louis, Mo.).

B. LC-MS/MS of Patient Sample Pools

To identify all CLIC and TPM isoforms that could be detected in ovariancancer patient sera, label-free comparisons of pooled sera wereperformed. One pool of serum from benign patients and three pools ofadvanced ovarian cancer patient serum samples were made. Serum poolswere immunodepleted, separated on a 1D SDS gel for 4 cm, sliced into 40fractions, and digested with trypsin (sequencing-grade, modified trypsinfrom Promega, Madison, Wis.) as described in Tang et al, 2012, citedabove and incorporated herein by reference). Tryptic digests wereanalyzed using an LTQ Orbitrap XL mass spectrometer (Thermo Scientific,Waltham, Mass.) connected to a NanoACQUITY UPLC system (Waters, Milford,Mass.). Peptides were eluted at 200 nL/min using a 229 min gradientconsisting of 5-28% B over 168 min, 28-50% B over 51.5 min, 50-80% Bover 5 min, 80% B for 4.5 min, before returning to 5% B over 0.5 min. Ashort blank gradient was run before injecting the next sample. The massspectrometer was set to scan m/z from 400 to 2000. The full MS scan wascollected at 60,000 resolution in the Orbitrap in profile mode followedby data-dependent MS/MS scans on the six-most-abundant ions exceeding aminimum threshold of 1000, collected in the linear trap. Monoisotopicprecursor selection was enabled and charge-state screening was enabledto reject z=1 ions. Ions subjected to MS/MS were excluded from repeatedanalysis for 60 s.

C. Data Processing

MS/MS spectra were extracted and searched using the SEQUEST algorithm(v. 28, rev. 13, University of Washington, Seattle, Wash.) in Bioworks(v. 3.3.1, Thermo Scientific) against the human UniRef100 proteinsequence database (v. June 2011) plus common contaminants. A decoydatabase was produced by reversing the protein sequence of each databaseentry, and the entire reversed database was appended in front of theforward database. Spectra were searched with a partial trypticconstraint with up to two missed cleavages, 100 ppm precursor masstolerance, 1 Da fragment ion mass tolerance, static modification of Cys(+57.0215 Da), and variable modification of Methionine (+15.9949 Da).Common or shared protein lists were created using DTASelect (v. 2.0,licensed from Scripps Research Institute, La Jolla, Calif.).

The following filters were applied: remove proteins that are subsets ofothers, full tryptic constraint, a minimum of two peptides, massaccuracy≦10 ppm, and ΔCn≧0.05 (Wang, H et al., J Proteome Res 2011, 10,(11), 4993-5005). The peptide false discovery rate in the resultingdataset was less than 1%. Quantitative comparisons of all detected CLICand TPM (related proteins from different genes and isoforms of a singlegene) across serum pools were performed using Rosetta Elucidatorsoftware to compare peptide signal intensities in full MS scans.Retention time alignment, feature identification (discrete ion signals),feature extraction, and protein identifications were performed by theElucidator system as previously described in Tang et al, 2012, Tang, etal, 2011; and Beer et al, 2011, all cited above.

D. Label-Free MRM Analysis

MRM experiments were performed on a 5500 QTRAP hybrid triplequadrupole/linear ion trap mass spectrometer (AB SCIEX, Foster City,Calif.) interfaced with a NanoACQUITY UPLC system. Eight μL of trypticdigests were injected using the partial loop injection mode onto a UPLCSymmetry trap column (180 μm i.d.×2 cm packed with 5 μm C18 resin;Waters) and then separated by RP-HPLC on a BEH C18 nanocapillaryanalytical column (75 μm i.d.×25 cm, 1.7 μm particle size; Waters) at45° C. Chromatography was performed with Solvent A, which containedMilli-Q water with 0.1% formic acid, and Solvent B, which containedHPLC-grade acetonitrile (Thomas Scientific, Swedesboro, N.J.) and 0.1%formic acid. Peptides were eluted at 400 nL/min for 5-35% B over 24 min,35% B for 3 min before returning to 5% B in 0.5 min.

To minimize sample carryover, a fast blank gradient was run between eachsample. An identical reference sample was run at the beginning of eachset of samples and was used to normalize variation in MRM signals causedby changes in performance of the HPLC, reverse phase column or massspectrometer. MRM data were acquired with a spray voltage of 3300 V,curtain gas of 20 p.s.i., nebulizer gas of 10 p.s.i., interface heatertemperature of 150° C., and a pause time of 3 ms. Multiple MRMtransitions were monitored using unit resolution in both Q1 and Q3quadrupoles to maximize specificity. Scheduled MRM was used to reducethe number of concurrent transitions and maximize the dwell time foreach transition. The detection window was set at 3 min, and the targetscan time was set at 1.8 s. Data analyses were performed usingMultiQuant version 2.1 software (AB SCIEX). The most abundant transitionfor each peptide was used for quantitation unless interference from thematrix was observed. In these cases, another transition free ofinterference was chosen for quantitation.

E. Statistical Analyses

Serum levels of candidate biomarkers were compared across patient groupsusing the Mann-Whitney test, and Bonferroni-adjusted P-values werereported in scatter plots. Results were considered statisticallysignificant if the Bonferroni-adjusted P-value of the test was less than0.05. Spearman's correlation coefficients were calculated to examinecorrelations among all tested tropomyosin peptides. For each candidatebiomarker, a receiver operator characteristic (ROC) curve was generatedand the area under the curve was calculated to reflect biomarkerspecific potential sensitivity and specificity for distinguishingnon-cancer and cancer patients.

Example 2 Ambiguities in Identification of EOC Candidate Biomarkers andIsoforms from Analysis of Xenograft Mouse Serum

We previously identified 106 human proteins with at least two peptidesfrom the serum of a xenograft mouse model of human ovarian endometrioidcancer (TOV-112D tumors) using a gel based, multidimensional proteinprofiling strategy.²¹ In that study, GeLC-MRM quantitation of severalcandidate biomarkers in the 20-55 kDa region showed that CLIC1, PRDX6,and the mature form of CTSD were significantly elevated in ovariancancer patients compared with noncancer (normal and benign disease)individuals.

Tropomyosin 1 (TPM1) isoform 6 was initially identified as a humanprotein in the xenograft mouse serum based upon the detection of twohuman-specific peptides and four peptides common to human and mouse.Changes of species specificity due to database updates was not awidespread problem, because reexamination of the species specificity ofour previously identified putative human peptides revealed that onlyseven peptides (approximately 1%) were identical to new mouse entries inthe 2011 database. Since both human and mouse TPM1 are also highlyhomologous (98 to 99% identical, depending on specific isoforms used forcomparison), it is extremely challenging to distinguish between the twospecies.

The confident identification of specific biomarkers proteins withinproteins families is often ambiguous when using shotgun proteomics,because protease digestion destroys the connectivity between proteinsand detected peptides. Inferring the correct peptide-protein associationis often confounded by the presence of shared peptides from homologousproteins that are not isoforms from the same gene, alternative splicevariants, or redundant database entries (Nesvizhskii, A. I.; Aebersold,R., Mol Cell Proteomics 2005, 4, (10), 1419-40). But for MRMquantitation, it is important to accurately determine thepeptide-protein relationship to ensure the correct form of a protein isbeing quantitated.

In order to determine all potential peptide-protein associations for theobserved TPM peptides, each peptide identified in the xenograft mousewas searched against the human UniProtKB database (February, 2012) usingthe BLAST algorithm. All database entries containing the peptidesequence were identified and redundant entries were manually removed.When available, gene names associated with each database entry were alsoextracted (see Table 2).

TABLE 2Peptides identified in TOV-112D xenograft mouse serum and human serum pools# Unique Database Sequence^(a) SEQ ID NO Entries GeneTPM1 Peptides Identified in Xenograft Mouse Serum ETAEADVASLN^(b)aa43-53 of SEQ ID NO. 6  6 TPM1 SLQEQADAAEER^(b)aa16-27 of SEQ ID NO. 10  7 TPM1 (K)LVIIESDLER^(b),aa132-142 of SEQ ID NO: 10 18 TPM1 MEIQEIQLK aa105-113 of SEQ ID NO. 638 TPM1, TPM3 IQLVEEELDR^(b), aa43-53 of SEQ ID NO. 6 43TPM1, TPM2, TPM3, TPM4 TPM1 Peptides Detected in Patient Serum PoolsETAEADVASLNR^(b) aa43-54 of SEQ ID NO. 6  6 TPM1 SLQEQADAAEER^(b),aa16-27 of SEQ ID NO. 10  7 TPM1 (K)LVIIESDLER^(b)aa132-142 of SEQ ID NO. 6 18 TPM1 MEIQEIQLK aa105-113 of SEQ ID NO. 6 38TPM1, TPM3 (R)IQLVEEELDR^(b) aa55-65 of SEQ ID NO. 6 43TPM1, TPM2, TPM3, TPM4 AELSEGQVR^(b) aa147-155 of SEQ ID NO: 10 10 TPM1YEEEIK^(c) aa185-190 of SEQ ID NO. 6 57 TPM1, TPM2, TPM3, TPM4ATDAEADVASLNR^(c,d) 17 19 TPM1, TPM2TPM2 Peptides Detected in Patient Serum Pools (K)LVILEGELER^(b) 18 11TPM2, TPM4 (R)IQLVEEELDR^(b) aa55-65 of SEQ ID NO: 10 43TPM1, TPM2, TPM3, TPM4 ATDAEADVASLNR^(c) 17 19 TPM1, TPM2 MELQEMQLK 20 8 TPM2 SLMASEEEYSTK 21  3 TPM2 YEEEIK^(c), aa185-190 of SEQ ID NO. 6 57TPM1, TPM2, TPM3, TPM4 EDKYEEEIK 22 31 TPM2, TPM3, TPM4 CGDLEEELK^(c) 23 6 TPM2, TPM4 TPM3 Peptides Detected in Patient Serum Pools(K)IQVLQQQADDAEER^(b) 24 16 TPM3 (R)IQLVEEELDR^(b),aa55-65 of SEQ ID NO: 10 43 TPM1, TPM2, TPM3, TPM4 MELQEIQLK 25 38TPM1, TPM3 (K)LVIIEGDLER 26 19 TPM3 HIAEEADR 27 22 TPM3, TPM4MLDQTLLDLNEM 28 12 TPM3 YEEEIK^(c), aa185-190 of SEQ ID NO. 6 57TPM1, TPM2, TPM3, TPM4 EDKYEEEIK 22 31 TPM2, TPM3, TPM4 CLSAAEEK 29 14TPM3 AADAEAEVASLNR 30  1 TPM3TPM4 Peptides Detected in Patient Serum Pools (K)IQALQQQADEAEDR^(b) 31 3 TPM4 (K)LVILEGELER^(b) 18 11 TPM2, TPM4 EENVGLHQTLDQTLNEL 32  4 TPM4NCI AEGDVAALNR^(b) 13  4 TPM4 MEIQEMQLK 33  2 TPM4 YSEKEDKYEEEIK 34  4TPM4 CGDLEEELK^(c) 23  6 TPM2, TPM4 (R)IQLVEEELDR^(b),aa55-65 of SEQ ID NO: 10 43 TPM1, TPM2, TPM3, TPM4 HIAEEADR 27 22TPM3, TPM4 EKAEGDVAALNR 35  3 TPM4 YEEEIK^(c), aa185-190 of SEQ ID NO. 657 TPM1, TPM2, TPM3, TPM4 EDKYEEEIK 22 31 TPM2, TPM3, TPM4 TIDDLEEK 36 4 TPM4 ASDAEGDVAALNR 37  1 TPM4 NOTES ^(a)Detected peptides shared bymultiple gene products are listed under all matching proteins.Underlined sequences were identified as human in the UniProtKB 2007database, but also matched a mouse homolog in a later database^(b)Peptide targeted for MRM. Successful quantitation. ^(c)Peptidetargeted for MRM. Unsuccessful quantitation. ^(d)This peptide is notpresent in the group of TPM1 isoforms (FIG. 3) defined by peptidesidentified from the xenograft mouse serum.

These peptides, i.e., KLVIIESDLE, aa132-142 of SEQ ID NO: 10,LVIIESDLER, aa133-142 of SEQ ID NO: 10; M*EIQEIQLK, aa105-113 of SEQ IDNO. 6; ETAEADVASLNR, aa43-54 of SEQ ID NO. 6; IQLVEEELDR, aa 43-53 ofSEQ ID NO:6; and SLQEQADAAEER, aa16-27 of SEQ ID NO: 10, show a greatdegree of ambiguity in peptide-protein association due to the largenumber of TPM family members and the number of known isoforms of severalof the family members. Tropomyosin is encoded by four genes (TPM1 toTPM4), and each gene can further generate multiple isoforms by the useof alternative promoters and/or alternative RNA splicing. More than 40distinct TPM family proteins and isoforms have been reported invertebrates (Gunning, P. W et al, Trends Cell Biol 2005, 15, (6),333-41; Choi, C et al. J Struct Biol 2012, 177, (1), 63-9). The TPM1peptides identified from the xenograft model were initially assigned toTPM1 isoform 6 (Q7Z6L8) using the parsimony principle to explain all theidentified peptides. While BLAST indicates TPM1 is present, the exactTPM1 isoform cannot be ascertained, and the presence of the relatedproteins, TPM2, TPM3, or TPM4 or the isoforms thereof, cannot beexcluded and should be considered.

Example 3 Biomarker Proteins Detectable in Patient Serum Pools thatCorrelate with Eoc

A. Tropomyosin Family Biomarkers

The tropomyosins are a family of actin filament-binding proteins thathave a well-defined central role in regulating muscle contraction andcytoskeletal organization in non-muscle cells. Decreased expression ofspecific tropomyosin isoforms is commonly associated with thetransformed phenotype and has been reported for cancer cells andtissues, including EOC (Helfinan, D. M. et al, Adv Exp Med Biol 2008,644, 124-31; Raval, G. N. et al, Oncogene 2003, 22, (40), 6194-203 andChow, S. N et al, Eur J Gynaecol Oncol 2010, 31, (1), 55-62). Theserological level of tropomyosin is not well studied, although highplasma levels of TPM4 have been associated with asbestos exposure, andincreased levels of tropomyosin serum antibodies were observed incolorectal cancer patients.

To determine which TPM family protein or isoform is/are detectable inovarian cancer patient serum, we used an ovarian patient serum proteindataset from in-depth GeLC-MS/MS analysis of the 20-55 kDa region of onebenign and three different late-stage ovarian cancer patientimmunoaffinity depleted serum pools.

In the case of TPM1, one new TPM1-specific peptide and two sharedpeptides were discovered in the patient serum in addition to previouslyidentified TPM1 isoform peptides from the xenograft mouse serum (SeeFIG. 3, Table 1 and Table 3). Table 3 shows high confidence biomarkerproteins/peptides detected in patient sera.

TABLE 3 (Part 1) Seq. Spectrum # Uniprot ID Name Description Count Count1 O00299 CLIC1 Chloride intracellular 12  67 channel protein 1 1a Q53FB0CLIC1 Chloride intracellular 12  67 channel 1 variant 2 Q9Y696 CLIC4Chloride intracellular 10  24 channel protein 4 3a Q1ZYL5 TPM1Tropomyosin 1 alpha variant  9  94 6 3b B7Z596 cDNA FLJ55130, highly  9 94 similar to Rattus norvegicus tropomyosin 1, alpha(Tpm1), transcript variant 8, mRNA 4a UR1H_P07951 TPM2Tropomyosin beta chain  9 125 4b UR1H_Q5TCU3 TPM2 Tropomyosin 2 (beta) 9 125 4c UR1H_Q5TCU8 TPM2 Tropomyosin 2 (beta)  9 125 5a P07951-2 TPM2Isoform 2 of Tropomyosin  9 107 beta chain 5b A7XZE4 TPM2Beta Troppomyosin isoform  9 107 6 P06753 TPM3 Tropomyosin alpha-3 chain 9  77 7a Q5VU59 TPM3 Tropomyosin-3 12 112 7b Q5HYB6 TPM3 DKFZp686J137212 112 7c B2RDE1 TPM3 cDNA, FLJ96568 12 112 7d E2RB38 TPM3Uncharacterized protein 12 112 8 P67936 TPM4 Tropomyosin alpha-4 chain16 243 9 P67936-2 TPM4 Isoform 2 of Tropomyosin 13 149 alpha-4 chain(Part 2) Seq # Coverage MolWt pI ObsM + H+ Ppm z XCorr DeltCN SpR 1 67.726923 5.2 1932.9794 3.1 2 4.4164 0.4663 1 1a 67.7 27015 5.2 1281.67 1.32 4.0828 0.3965 1 1844.9752 0 2 4.3773 0.5176 1 2992.3948 1.4 3 5.75640.546 1 1328.6461 1.6 2 3.3785 0.4031 1 1095.6344 0.2 2 2.4544 0.1432 11065.6228 5.9 2 2.3876 0.0719 1 2573.1345 4.3 2 3.7106 0.3711 1 2061.0721.7 3 3.2149 0.2172 2 1572.8121 2 2 2.8708 0.1944 1 1078.5076 −2.1 22.2701 0.16 6 957.4788 −0.1 2 2.0529 0.062 13 2 52.6 28772 5.6 1264.71580.9 2 3.8998 0.3919 1 2595.1702 1.8 3 4.4279 0.3832 1 1385.6783 1.1 33.8417 0.2936 1 1223.6191 3.1 2 3.0191 0.2578 1 1079.6346 2.3 2 2.660.1121 1 1313.5336 1.7 2 2.695 0.2585 1 1602.8118 2.2 2 2.3922 0.2966 11966.9231 −0.9 2 2.6045 0.239 1 1588.8486 1.1 3 2.2303 0.1033 811533.7018 1 2 1.9489 0.0741 34 3a 28.6 28509 4.8 1243.6528 0 2 3.65740.283 1 3b 25.5 31753 4.9 1346.6198 1.1 2 3.8906 0.3488 1 1186.6775 8.32 2.8985 0.1839 1 1147.6036 0.8 2 3.3535 0.1265 1 1275.6222 3.7 2 3.6960.3306 1 1314.7639 0.9 3 3.8858 0.3198 1 988.5064 0.6 2 2.6379 0.1755 11399.7528 −0.9 3 3.3619 0.2539 11 810.3899 2.3 2 1.8363 0.0982 13 4a21.8 32851  4.7 1298.7697 1.4 3 4.5416 0.2826 1 4b 21.8 32815  4.71170.6774 3.9 2 3.6614 0.2594 1 4c 19.3 36747  4.8 1243.6528 0 2 3.65740.283 1 1332.6372 −1.3 2 3.5477 0.261 1 1181.5554 1.2 2 2.9144 0.1323 31092.4814 −5.8 2 3.0957 0.1527 2 1399.7528 −0.9 3 3.3619 0.2539 11810.3899 2.3 2 1.8363 0.0982 13 1182.5557 2.7 2 2.3489 0.1214 35 5a 22.932990  4.7 1298.7697 1.4 3 4.5416 0.2826 1 5b 22.9 33026  4.7 1170.67743.9 2 3.6614 0.2594 1 1243.6528 0 2 3.6574 0.283 1 1332.6372 −1.3 23.5477 0.261 1 1181.5554 1.2 2 2.9144 0.1323 3 1399.7528 −0.9 3 3.36190.2539 11 1390.6013 −2.1 2 2.326 0.3071 1 810.3899 2.3 2 1.8363 0.098213 1182.5557 2.7 2 2.3489 0.1214 35 6 21.5 32819   4.7 1243.6528 0 23.6574 0.283 1 1147.6036 0.8 2 3.3535 0.1265 1 1284.7534 0.9 3 3.55490.2933 1 1156.6655 7.2 2 3.2166 0.1613 2 1399.7528 −0.9 3 3.3619 0.253911 940.4491 0.8 2 2.3445 0.1573 1 1316.6499 4.4 3 3.194 0.0726 2810.3899 2.3 2 1.8363 0.0982 13 1182.5557 2.7 2 2.3489 0.1214 35 7a 35.827175 4.8 1642.8102 4.3 2 5.1395 0.4843 1 7b 35.8 27176 4.7 1770.897−0.6 3 4.6197 0.4128 1 7c 33.5 29018 4.8 1243.6528 0 2 3.6574 0.283 1 7d33.5 29033 4.8 1147.6036 0.8 2 3.3535 0.1265 1 1284.7534 0.9 3 3.55490.2933 1 1156.6655 7.2 2 3.2166 0.1613 2 1399.7528 −0.9 3 3.3619 0.253911 940.4491 0.8 2 2.3445 0.1573 1 1467.6772 4.6 2 2.864 0.2482 2810.3899 2.3 2 1.8363 0.0982 13 1182.5557 2.7 2 2.3489 0.1214 35907.4202 1.4 2 1.7519 0.0886 33 8 46.8 28522   4.7 1614.7726 0.5 24.8416 0.5205 1 1742.8723 3.2 3 5.3142 0.4742 1 1298.7697 1.4 3 4.54160.2826 1 2340.1206 3 2 5.717 0.2539 1 1170.6774 3.9 2 3.6614 0.2594 11243.6528 0 2 3.6574 0.283 1 1015.5159 −0.8 2 3.1643 0.3492 1 1181.55541.2 2 2.9144 0.1323 3 1689.7849 −0.3 3 3.9281 0.2333 1 1092.4814 −5.8 23.0957 0.1527 2 1399.7528 −0.9 3 3.3619 0.2539 11 940.4491 0.8 2 2.34450.1573 1 1272.659 3.7 3 3.0739 0.2454 11 810.3899 2.3 2 1.8363 0.0982 131182.5557 2.7 2 2.3489 0.1214 35 962.4657 −2.1 2 1.5493 0.0793 90 9 35.932723 4.7 1298.7697 1.4 3 4.5416 0.2826 1 2340.1206 3 2 5.717 0.2539 11170.6774 3.9 2 3.6614 0.2594 1 1243.6528 0 2 3.6574 0.283 1 1181.55541.2 2 2.9144 0.1323 3 1689.7849 −0.3 3 3.9281 0.2333 1 1092.4814 −5.8 23.0957 0.1527 2 1399.7528 −0.9 3 3.3619 0.2539 11 1288.6102 −1.9 22.8922 0.1827 1 940.4491 0.8 2 2.3445 0.1573 1 810.3899 2.3 2 1.83630.0982 13 1182.5557 2.7 2 2.3489 0.1214 35 962.4657 −2.1 2 1.5493 0.079390 (Part 3) No Sequence^(b) SEQ ID NO. 1a K.FLDGNELTLADCNLLPK.L 38 1bK.GVTFNVTTVDTK.R 39 K.LAALNPESNTAGLDIF AK.F 40K.VLDNYLTSPLPEEVDETSAEDEGVSQR.K 41 K.NSNPALNDNLEK.G 42 K.LHIVQVVCK.K 43R.LFM*VLWLK.G 44 R.EEFASTCPDDEEIELAYEQVAK.A 45 R.KFLDGNELTLADCNLLPK.L 46K.IEEFLEAVLCPPR.Y 47 K.IGNCPFSQR.L 48 R.YLSNAYAR.E 49 2 KGVVFSVTTVDLK.Raa48-61 of SEQ ID NO: 1 K.LDEYLNSPLPDEIDENSM*EDIK.Faa150-173 of SEQ ID NO: 1 K.NSRPEANEALER.G aa130-143 of SEQ ID NO: 1K.EVEIAYSDVAK.R aa237-250 of SEQ ID NO: 1 R.LFM*ILWLK.G^(c)aa40-49 of SEQ ID NO: 1 R.DEFTNTCPSDK.E aa227-239 of SEQ ID NO: 1K.IEEFLEEVLCPPK.Y aa90-104 of SEQ ID NO: 1 K.FLDGNEM*TLADCNLLPK.Laa177-195 of SEQ ID NO: 1 K.EEDKEPLIELFVK.A aa11-25 of SEQ ID NO: 1K.HPESNTAGM*DIFAK.F aa110-125 of SEQ ID NO: 1 3a R.IQLVEEELDR.A 50 3bR.SLQEQADAAEER.A aa15-28 of SEQ ID NO: 10 K.LVIIESDLER.Aaa132-143 of SEQ ID NO: 10 K.M*EIQEIQLK.E aa104-114 of SEQ ID NO. 6R.ETAEADVASLNR.R aa42-55 of SEQ ID NO. 6 R.KLVIIESDLER.Aaa131-143 of SEQ ID NO: 10 R.AELSEGQVR.Q aa147-156 of SEQ ID NO: 10R.RIQLVEEELDR.A 51 R.YEEEIK.V aa184-191 of SEQ ID NO. 6 4aR.KLVILEGELER.S 19 4b K.LVILEGELER.S 52 4c R.IQLVEEELDR.A 50K.ATDAEADVASLNR.R 53 K.M*ELQEM*QLK.E 54 K.CGDLEEELK.I 55 R.RIQLVEEELDR.A51 K.YEEEIK.L 56 K.EDKYEEEIK.L 58 5a R.KLVILEGELER.S 19 5bK.LVILEGELER.S 52 R.IQLVEEELDR.A 50 K.ATDAEADVASLNR.R 53 K.M*ELQEM*QLK.E54 R.RIQLVEEELDR.A 51 K.SLM*ASEEEYSTK.E 57 K.YEEEIK.L 56 K.EDKYEEEIK.L58 6 R.IQLVEEELDR.A 50 KM*ELQEIQLK.E 59 R.KLVIIEGDLER.T 60K.LVIIEGDLER.T 61 R.RIQLVEEELDR.A 51 K.HIAEEADR.K 62 K.AADAEAEVASLNR.R63 K.YEEEIK.I 64 K.EDKYEEEIK.I 65 7a KIQVLQQQADDAEERA 66 7bRKIQVLQQQADDAEER.A 67 7c RIQLVEEELDR.A 50 7d KM*ELQEIQLKE 68RKLVIIEGDLER T 60 K.LVIIEGDLER.T 61 RRIQLVEEELDRA 50 KHIAEEADRK 62RM*LDQTLLDLNEM* 69 KYEEEIKI 64 K.EDKYEEEIK.I 65 KCLSAAEEKY 70 8K.IQALQQQADEAEDRA 71 RKIQALQQQADEAEDR.A 72 RKLVILEGELERA 73KEENVGLHQTLDQTLNELNCL 74 KLVILEGELERA aa2-13 of SEQ ID NO: 73RIQLVEEELDRA aa2-14 of SEQ ID NO: 50 KAEGDVAALNR.Raa3-14 of SEQ ID NO: 75 K.M*EIQEM*QLK.E 76 K.YSEKEDKYEEEIK.L 77K.CGDLEEELKN 78 R.RIQLVEEELDRA 50 K.HIAEEADRK 62 REKAEGDVAALNR.R 75K.YEEEIK.L 56 K.EDKYEEEIK.L 58 K.TIDDLEEK.L 79 9 RKLVILEGELERA 73KEENVGLHQTLDQTLNELNCL 74 K.LVILEGELER.A aa2-13 of SEQ ID NO: 73R.IQLVEEELDR.A 50 K.M*EIQEM*QLK.E 76 K.YSEKEDKYEEEIK.L 77 K.CGDLEEELKN78 R.RIQLVEEELDR.A 51 KASDAEGDVAALNR.R 80 K.HIAEEADR.K 62 K.YEEEIK.L 56K.EDKYEEEIK.L 58 K.TIDDLEEK.L 79 ^(a)All peptides mapping to listedprotein isoforms are shown, i.e., peptides shared by multiple proteinisoforms are listed for each of these multiple proteins. ^(b)Differentforms of the same peptide (charge states and methionine oxidation) werecollapsed and displayed as a single peptide. M* indicates methionineoxidation. ^(c)This peptide is also present in CLIC2, CLIC5, and CLI6.

Based on the newly identified AELSEGQVR, AA147-155 of SEQ ID NO: 10,peptide, all observed peptides were contained within two TPM1 isoforms,TPM1 variant 6 (Q1ZYL5) or B7Z596. These two sequences share 80%identity and differ from each other at the C-terminus Distinguishingbetween these isoforms was not feasible in this study due to theinability to detect any isoform-specific C-terminal peptides. Althoughno other TPM1 isoforms or TPM family members were conclusivelyidentified in human serum, their presence cannot be ruled out. But thefailure to detect any unique peptides to other TPM family members orTPM1 isoforms suggests they are either not present or are present inmuch lower abundance in human serum.

The TPM family proteins and/or their isoforms identified in the patientsera were quantitated by summing MS intensities for all peptides uniqueto a specific gene product (see FIGS. 1A through 1D). There was evidenceof protein products for all four TPM genes and the expressed relatedgene products showed elevated levels in EOC.

B. Chloride Ion Channel Protein Biomarkers

Two CLIC family proteins were identified in the patient sera and werequantitated by summing MS intensities for all peptides unique to aspecific gene product (see FIGS. 1E and 1F). The previously identifiedCLIC1 was confirmed to be both detected and elevated in ovarian cancerpatient serum compared to benign disease.

Another CLIC family member protein CLIC4 was newly identified in theovarian cancer patient sera. CLIC4 is a multifunctional protein that hasbeen shown to be highly expressed in ovarian cancer stroma and may playan important role in cancer development (Yao, Q et al., Oncol Rep 2009,22, (3), 541-8; Shukla, A.; Yuspa, S. H., Nucleus 2010, 1, (2), 144-9).CLIC4 was detected by nine specific peptides and showed elevated levelsin ovarian cancer patient sera, suggesting that it was an EOC biomarker.The observation of CLIC4 in ovarian cancer patient sera raised thequestion as to why human CLIC1 had been previously identified in thexenograft mouse serum, but CLIC4 had not been detected.

Examination of this data showed that CLIC4 was identified by fourpeptides, all peptides identical to mouse sequences, i.e.,K.GVVFSVTTVDLK.R, aa48-61 of SEQ ID NO: 1; K.HPESNTAGM*DIFAK.F,aa110-125 of SEQ ID NO: 1; K.LDEYLNSPLPDEIDENSM*EDIK.F, aa150-173 of SEQID NO: 1; and R.KPADLQNLAPGTHPPFITFNSEVK.T, aa61-86 of SEQ ID NO: 1.This is not surprising, as the human and mouse CLIC4 sequences are 99%identical (See FIG. 2A and Table 4). While distinguishing between mouseand human CLIC4 is very difficult, this problem is limited to thexenograft mouse model. In contrast, distinguishing the different CLICfamily proteins, e.g., gene products, in human serum is morestraightforward, as the sequence homologies of the four CLIC genes withsimilar molecular weights are somewhat lower. Specifically, the two CLICfamily proteins detected, CLIC1 and CLIC4, share 67% identity. Hence,most CLIC peptides observed in the xenograft mouse serum and in patientserum pools were unique to either CLIC1 or CLIC4 (see FIG. 2B).

Example 4 Development of MRM Assays for Quantitation of CLIC4 and TPMIsoforms

CLIC and TPM biomarker protein levels in individual serum samples thatincluded control serum samples (six normal and nine benign) andlate-stage cancer samples (15 Stage III and 3 Stage IV) were determinedusing GeLC-MRM, essentially as previously described in Tang et al 2012,cited above. Peptides were selected based on their isoform specificityand signal intensity in MRM analysis using a 5500 QTRAP massspectrometer. Peptide candidates for MRM were derived from a combinationof the LCMS/MS analyses reported above and all human plasma/serumLC-MS/MS proteomic analyses that had been performed in this laboratory.

In the case of CLIC4, selection of MRM peptides was relativelystraightforward because no major isoform issues were encountered withthe identified peptides (FIG. 2B). Inclusion of peptides identified fromother serum proteome analyses allowed selection of peptides with thestrongest MRM signal. For example, the CLIC4 peptide, YLTNAYSR,aa220-227 of SEQ ID NO: 1, was found to produce a stronger MRM signalthan some of the peptides discovered in this analysis and was thereforeused for MRM quantitation (See Tables 4 and 5). Table 4 shows MRM peakareas for CLIC4 peptides, which have been normalized and averaged inindividual samples as an indication of the protein abundance level.

TABLE 4 CLIC4 CLIC4 CLIC4 NSRPEANEALER, YLTNAYSR EVEIAYSDVAK, CLIC4 aa131-142 of aa220-227 of aa 139-249 of Norm & Sample # SEQ ID NO: 1 SEQID NO: 1 SEQ ID NO: 1 Avg Normal WCS02 71938 75784 5917 0.171 NormalWCS04 71504 94424 13193 0.214 Normal WCS12 58303 67969 5269 0.146 NormalWCS13 92896 92885 21819 0.268 Normal WCS14 133409 130748 38618 0.410Normal WCSI5 90830 100865 28662 0.298 Benign B23 106848 144779 285060.355 Benign B25 93943 128049 23848 0.308 Benign B70 142389 164356 283630.412 Benign B77 180776 242906 75215 0.699 Benign B79 121455 16277839150 0.427 Benign B80 233045 287533 87063 0.841 Benign B81 125616150244 36499 0.411 Benign B82 162386 269199 41618 0.578 Benign B83120162 217288 50882 0.518 Cancer T455 361477 343172 106250 1.106 CancerT474 444226 449309 98203 1.264 Cancer T475 205961 249565 36565 0.590Cancer T476 120474 170584 22532 0.371 Cancer T478 236002 254455 935430.839 Cancer T482 380003 516406 80345 1.187 Cancer T536 443069 340463308048 1.942 Cancer T539 249682 306233 49728 0.739 Cancer T541 87267113303 17938 0.266 Cancer T543 107946 206527 44406 0.471 Cancer T55341$869 618499 114770 1.446 Cancer T556 959630 1244493 256878 3.143Cancer T557 266741 378642 60233 0.862 Cancer T577 176341 183183 417240.516 Cancer T600 360817 393236 87364 1.080 Cancer T602 167445 26609568203 0.679 Cancer T603 257545 473664 89399 1.045 Cancer T604 114903174142 46015 0.455 Cancer T455 361477 343172 106250 1.106

Table 5 shows MRM peak areas for CLIC1 peptides normalized and averagedin individual samples as an indication of the protein abundance level.

TABLE 5 CLIC1 CLIC1 CLIC1 GVTFNVTTVDTK, LAALNPESNTAG NSNPALNDNLEK, CLIC1aa38-49 of LDIFAK, aa96-113 aa120-131 of Norm & Sample # SEQ ID NO: 3 ofSEQ ID NO: 3 SEQ ID NO: 3 Avg Normal WCS02 112466 46620 67000 0.099Normal WCS04 201327 82090 148050 0.190 Normal WCS12 33884 30160 511000.054 Normal WCS13 28858 32810 30770 0.044 Normal WCS14 164134 155000173700 0.230 Normal WCS15 104943 85700 87400 0.128 Benign B23 143772113800 131900 0.179 Benign B25 105300 27596 58447 0.082 Benign B70210921 194300 220300 0.313 Benign B77 680255 387400 491200 0.704 BenignB79 452783 340500 393500 0.544 Benign B80 754653 168561 528925 0.623Benign B81 168894 56262 137171 0.159 Benign B82 1367992 623771 10399901.347 Benign B83 549811 153388 395116 0.476 Cancer T455 980711 546125697954 0.999 Cancer T474 819310 351580 518150 0.745 Cancer T475 286624205100 204300 0.318 Cancer T476 414920 306150 290460 0.463 Cancer T478485804 298830 183150 0.435 Cancer T482 2816690 2008424 2505141 3.373Cancer T536 1178896 964000 1143310 1.516 Cancer T539 651564 398980428100 0.667 Cancer T541 49917 27680 36000 0.051 Cancer T543 511810232600 314500 0.469 Cancer T553 1993369 1119800 1503310 2.076 CancerT556 2808408 2207100 2532200 3.472 Cancer T557 651252 444800 4408000.699 Cancer T577 346698 214500 277400 0.380 Cancer T600 567452 358500471000 0.633 Cancer T602 455781 280500 317400 0.476 Cancer T603 771899505560 620200 0.862 Cancer T604 280053 204600 316300 0.367

Selecting appropriate peptides for MRM quantitation of TPM1 isoforms andTPM family proteins in general, and TPM1 specifically, was morecomplicated due to the large number of TPM family members and isoforms.While TPM1 variant 6 (or isoform B7Z596) was clearly identified in thehuman serum samples, other TPM1 isoforms also could be present (See FIG.3). Therefore, AELSEGQVR, aa147-155 of SEQ ID NO: 10, which was specificto TPM1v6, and three other peptides shared by several TPM1 isoforms andother TPM family members were used for MRM quantitation (see Table 1,and Table 6). Table 6 shows MRM peak areas for TPM1 peptides normalizedand averaged in individual samples as an indication of the proteinabundance level.

TABLE 6 TPM1v6 TPM1v6 TPM1v6 TPM1v6 LVIIESDLER, AELSEGQVR ETAEADVAS LNR,SLQEQADA AEER, TPM1v6 aa133-142 of aa147-155 of aa43-54 of aa16-27 ofNorm& Sample # SEQ ID NO: 10 SEQ ID NO: 10 SEQ ID NO. 6 SEQ ID NO: 10Avg Normal WCS02 27232 74344 20979 11989 0.142 Normal WCS04 8305 8047713397 9571 0.089 Normal WCS12 2975 54062 9440 6603 0.056 Normal WCS134094 41958 0 4239 0.033 Normal WCS14 44163 149293 40975 35597 0.287Normal WCS15 11741 63033 15414 9777 0.093 Benign B23 47853 257616 5105438261 0.357 Benign B25 14674 123609 30139 23165 0.175 Benign B70 46999382365 66409 43960 0.440 Benign B77 101250 492555 105122 72337 0.713Benign B79 50274 251060 48504 38126 0.355 Benign B80 145616 872565306980 196336 1.542 Benign B81 45480 275283 60738 47356 0.395 Benign B8253860 368472 128792 84337 0.638 Benign B83 56220 197314 86720 627440.500 Cancer T455 67782 428888 90666 64820 0.583 Cancer T474 1818631122884 207117 179329 1.506 Cancer T475 50783 255305 45158 34454 0.344Cancer T476 119220 523437 163132 86059 0.885 Cancer T478 120774 500688118103 98305 0.832 Cancer T482 71530 313303 43307 31577 0.398 CancerT536 431057 1930569 410941 320850 2.941 Cancer T539 104008 533374 12359291636 0.806 Cancer T541 0 31284 0 0 0.012 Cancer T543 56020 257055 5784044155 0.397 Cancer T553 209330 1191620 294342 236825 1.854 Cancer T556279781 1873212 438823 361745 2.754 Cancer T557 140328 662386 164727117159 1.048 Cancer T577 25267 233927 50457 44460 0.316 Cancer T60069940 417227 96263 70731 0.605 Cancer T602 50571 210865 65650 518360.399 Cancer T603 234813 1190812 242276 167008 1.667 Cancer T604 57236423975 125785 80801 0.652

We also attempted to target TPM2, TPM3, and TPM4, as products of thesegenes were also identified in the GeLC-MS/MS analysis of patient serumpools (Table 3). Of course, as is typically the case, strong,consistent, interference-free MRM signals could not be obtained for alldesired peptides. The final MRM assay contained one peptide specific toTPM3, two peptides specific to TPM4, one peptide shared by TPM2 andTPM4, and one peptide shared by all four TPM genes (See Table 1 andTable 7). Table 7 shows MRM peak areas for TPM3 and TPM4 peptidesnormalized and averaged in individual samples as an indication of theprotein abundance level.

TABLE 7 TPM3 TPM4 TPM4 TPM4 IQVLQQQAD TPM3 IQALQQQ AEGDVA KLVILEG TPM4DAEER, Norm& ADEAEDR, ALNR, ELER, Norm& Sample # SEQ ID NO: 12 Avg SEQID NO: 14 SEQ ID NO: 13 SEQ ID NO: 18 Avg Normal WCS02 37607 0.116 66530657724 630468 0.110 Normal WCS04 62540 0.192 57080 425479 419663 0.079Normal WCS12 11573 0.036 13120 179765 212806 0.030 Normal WCS13 0 0.0000 70176 140492 0.012 Normal WCS14 44708 0.137 18371 173740 238804 0.034Normal WCS15 23374 0.072 21477 215144 266941 0.039 Benign B23 508260.156 92784 769719 861540 0.144 Benign B25 31580 0.097 64680 592048641548 0.106 Benign B70 215328 0.661 203602 1962777 1862029 0.331 BenignB77 205003 0.630 455168 3475540 3751568 0.660 Benign B79 113763 0.349173730 1258455 1590148 0.257 Benign B80 651126 2.000 1431956 106309689275897 1.913 Benign B81 135085 0.415 167413 269426 1290576 0.237 BenignB82 533252 1.638 85394 681075 841789 0.134 Benign B83 210727 0.647216592 1631897 1912271 0.320 Cancer T455 181883 0.559 329762 31982113703179 0.517 Cancer T474 385261 1.183 1033082 8868498 8154397 1.539Cancer T475 78718 0.242 138849 1355419 1412261 0.235 Cancer T476 2871080.882 740508 7091117 6861693 1.207 Cancer T478 163723 0.503 3270302662922 2613253 0.480 Cancer T482 114583 0.352 264911 2633676 27208940.453 Cancer T536 876097 2.691 1796201 16550308 16225609 2.863 CancerT539 249410 0.766 492600 3835421 3916627 0.711 Cancer T541 0 0.000 702247530 49422 0.009 Cancer T543 147454 0.453 143235 1017944 977404 0.191Cancer T553 909385 2.793 1389863 8780337 10176023 1.830 Cancer T5561070392 3.288 2182304 16019177 17806655 3.116 Cancer T557 394944 1.213729285 6244945 7007301 1.157 Cancer T577 66040 0.203 126311 872615987295 0.174 Cancer T600 191799 0.589 466198 4198007 6027998 0.842Cancer T602 69239 0.213 168912 1430173 1592082 0.265 Cancer T603 4770131.465 1191352 10077129 11834475 1.908 Cancer T604 197319 0.606 3462422351772 2126914 0.442

Table 8 shows MRM peak areas for the TPM common peptide normalized inindividual samples as an indication of the protein abundance level.

TABLE 8 TPM RIQLVEEELDR, TPM Sample # SEQ ID NO: 16 Norm & Avg NormalWCS02 716335 0.114 Normal WCS04 680639 0.108 Normal WCS12 273406 0.043Normal WCS13 121600 0.019 Normal WCS14 519755 0.083 Normal WCS15 4442690.071 Benign B23 1018792 0.162 Benign B25 784378 0.125 Benign B702396858 0.381 Benign B77 4183895 0.065 Benign B79 1122745 0.274 BenignB80 12378679 1.967 Benign B81 1384354 0.220 Benign B82 3515417 0.559Benign B83 2601924 0.413 Cancer T455 3561471 0.566 Cancer T474 93459621.485 Cancer T475 1536987 0.244 Cancer T476 7037409 1.118 Cancer T4783371869 0.536 Cancer T482 2992930 0.476 Cancer T536 17007129 2.702Cancer T539 4446598 0.706 Cancer T541 148179 0.024 Cancer T543 15749560.250 Cancer T553 13973974 2.220 Cancer TS56 19572892 3.110 Cancer T5576989658 1.111 Cancer T577 1159454 0.184 Cancer T600 5455926 0.867 CancerT602 1665550 0.265 Cancer T603 10720865 1.703 Cancer T604 2728471 0.434

Example 5 Quantitation of TPM and CLIC Isoforms in Patient Serum Samples

GeLC-MRM quantitation of the CLIC and TPM peptides and normalizedprotein values for individual patient samples are summarized in Tables3-8. TPM peptide amounts were graphically compared across all serumsamples as a first-level test of potential differences across patientsin peptides specific for certain isoforms and those shared by multipleisoforms both within the TPM1 isoform group and across related geneproducts (See Table 3).

Similarly, the protein levels (normalized and averaged peptides values)across patient samples were compared as shown in FIG. 4. All thepeptides monitored displayed similar quantitative profiles, althoughsome minor variations were observed in some samples (See FIG. 4 andTable 3). Spearman's rank correlation coefficient analysis showed thatall tropomyosin peptides analyzed here are highly correlated within eachpatient group with P-values<0.001 (data not shown), indicating a lack ofevidence that specific tropomyosins differ from other family members andisoforms in being able to distinguish between ovarian cancer, benigndisease, and normal donors. Also, if alternative TPM family proteinsshare some of the quantified peptides, their contribution is eitherminor or they track with the TPM proteins and isoforms quantitated here.Since the distribution of all tropomyosin peptides is similar, thepeptide (K)LVILEGELER SEQ ID NO: 18 that is shared between TPM2 and TPM4was assigned to TPM4 for the purpose of calculating the TPM4 proteinlevel. In addition, factor analysis shows that all the tropomyosinproteins analyzed here are measuring the same factor (data not shown),which is consistent with the similar quantitative profiles shown in FIG.4.

These data show that multiple TPM related proteins and isoforms thereofare present in human serum. At least in the current cohort, fluctuationsin abundance levels related to benign ovarian disease and ovarian cancerfor TPM family members and their isoforms appear to change in concert.However, it is possible that certain TPM isoforms may be more selectivefor specific clinical applications such as monitoring responses todifferent therapies or disease reoccurrence. Tropomyosin proteins arealso known to be modified by post-translational modifications such asacetylation and phosphorylation, and the influence ofpost-translationally modified forms in ovarian cancer diagnosis has notyet been tested.

GeLC-MRM quantitative results for CLIC1 and CLIC4 peptide and proteinlevels also are shown in Tables 6 and 5, respectively. As expected,since the same patient samples were used, CLIC1 results were similar,but not identical, to the previously reported results in Tang et al,2012, cited above, for this protein. There was some moderate variationbecause the two sets of label-free measurements were performed atdifferent times and on two different instruments. That is, previousanalyses were performed on an AB SCIEX 4000 QTRAP and the currentresults were from an AB SCIEX 5500 QTRAP instrument. The CLIC1measurements were repeated here to provide a direct comparison to thenewly identified biomarker CLIC4. For both CLIC proteins, all peptidesfrom the same protein showed similar distributions among the individualpatient samples (data not shown), indicating that the MRM signals usedfor quantitation were derived from the same protein and quantitation wasnot appreciably affected by interfering signals.

Example 6 CLIC and TPM Biomarkers can Distinguish EOC from Non-CancerCases

The capacities of the CLIC and TPM biomarkers to distinguish EOC caseswere assessed in several ways using the GeLC-MRM quantitation data (See,Tables 4-8). A two-way comparison between the non-cancer (normal andbenign) and cancer groups using the Mann-Whitney test showed that allisoforms could significantly distinguish (P<0.05) between cancer andnon-cancer (see Table 9).

TABLE 9 GeLC-MRM comparison of non-cancer (normal and benign) versuscancer sera UniProt Name Descriptive Name P-value^(a) O00299 CLIC1Chloride intracellular 0.004 channel protein 1 Q9Y696 CLIC4 Chlorideintracellular 0.0002 channel protein 4 Q1ZYL5 TPM1, Tropomyosin 1 alphavariant 6 0.0052 var. 6 Q5VU59 TPM3 Tropomyosin 3 0.0337 P67936 TPM4Tropomyosin alpha-4 chain 0.0021 NOTES ^(a)P-values are from theMann-Whitney test with Bonferroni adjustment

Based on the P-value, CLIC4 appeared to be the best biomarker indistinguishing cancer from non-cancer and TPM3 was the weakestbiomarker.

For further evaluation, the normal and benign samples were comparedseparately to the cancer group (FIG. 5). All protein isoforms coulddistinguish between normal donors and ovarian cancer patients at ahighly significant level (P<0.0015). However, CLIC4 was the onlybiomarker that showed a significant difference between benign diseaseand EOC.

The TPM isoforms did not show a statistically significant differencebetween benign disease and cancer primarily because a single benignsample (B80) had a much higher abundance level for all TPM isoforms thanother samples in that group. Future analysis of larger cohorts willallow us to more definitively identify which proteins can reliablydistinguish benign disease from ovarian cancer.

Although benign ovarian tumors and ovarian cancer appear to be verydifferent diseases at a genetic level, a major challenge at the initialdiagnosis stage in the clinic is to distinguish benign ovarianconditions from malignant ones.

To evaluate the potential diagnostic efficacy for each of theseproteins, receiver operating characteristic (ROC) curve analyses wereperformed on the non-cancer and cancer groups (See FIG. 6). Consistentwith the Mann-Whitney test, CLIC4 showed the largest area under thecurve (AUC) and TPM3 showed the lowest area. It is anticipated thatlarger patient cohorts may support the use of CLIC4 and tropomyosinfamily proteins or isoforms, when used with other biomarkerssimultaneously and in combination may outperform use of a singlebiomarker for detection and clinical monitoring of EOC.

To our knowledge, the plasma levels of CLIC4 and tropomyosin in ovariancancer patients have not been reported previously.

It is anticipated that testing of the biomarkers and biomarkers setsdescribed herein in larger cohorts of patients collected from differentsites, longitudinal prediagnostic blood specimens, and specimenscollected throughout therapeutic treatment will demonstrate resultsconsistent with the above.

Each and every patent, patent application, and publication, includingU.S. provisional patent application No. 61/709,695, the publicationslisted herein, and publically available peptide sequences, citedthroughout the disclosure, is expressly incorporated herein by referencein its entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention are devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims include such embodiments and equivalent variations.

PUBLICATIONS

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The invention claimed is:
 1. A diagnostic reagent consisting of: (a) aligand capable of specifically complexing with, binding to, orquantitatively detecting or identifying the biomarker chlorideintracellular channel protein 4 (CLIC4) or an isoform, pro-form,modified molecular form, posttranslational modification, or uniquepeptide fragment or unique nucleic acid fragment thereof; (b) a ligandcapable of specifically complexing with, binding to, or quantitativelydetecting or identifying the biomarker chloride intracellular channelprotein 1 (CLIC1) or an isoform, pro-form, modified molecular form,posttranslational modification, or unique peptide fragment or uniquenucleic acid fragment thereof; and (c) a ligand capable of specificallycomplexing with, binding to, or quantitatively detecting or identifyingthe biomarker cathepsin D-30 (CTSD-30) or an isoform, pro-form, modifiedmolecular form, posttranslational modification, or unique peptidefragment or unique nucleic acid fragment thereof; wherein at least oneof the ligands is attached to a detectable label or immobilized on asubstrate.
 2. The diagnostic reagent according to claim 1, wherein thebiomarker CLIC4 is Uniprot ID no. Z9Y696 or a peptide fragment thereof.3. The diagnostic reagent according to claim 1, wherein the CLIC4peptide fragment comprises: (a) GVVFSVTTVDLK, aa 49-60 of SEQ ID NO: 1;(b) LDEYLNSPLPDEIDENSMEDIK, aa151-172 of SEQ ID NO: 1; (c)NSRPEANEALER, aa 131-142 of SEQ ID NO: 1; (d)YLTNAYSR, aa 20-27 of SEQ ID NO: 1; (d)EVEIAYSDVAK, aa 139-249 of SEQ ID NO: 1; (e)LFMILWLK, aa 41-48 of SEQ ID NO: 1; (f)DEFTNTCPSDK, aa 228-238 of SEQ ID NO: 1; (g)IEEFLEEVLCPPK, aa 91-103 of SEQ ID NO: 1; (h)FLDGNEMTLADCNLLPK, aa 178-194 of SEQ ID NO: 1; (i)EEDKEPLIELFVK, aa 12-24 of SEQ ID NO: 1; or (j)HPESNTAGMDIFAK, aa 111-124 of SEQ ID NO: 1;

wherein in each sequence where methionine occurs, it is in the oxidizedor unoxidized form.
 4. The diagnostic reagent or device according toclaim 1, wherein the CLIC1 peptide fragment comprises: (a)GVTFNVTTVDTK, aa38-49 of SEQ ID NO: 3; (b)LAALNPESNTAGLDIFAK, aa96-113 of SEQ ID NO: 3; or (c)NSNPALNDNLEK, aa120-131 of SEQ ID NO:
 3.


5. The diagnostic reagent according to claim 1, wherein any of saidligands comprises an antibody or fragment of an antibody, an antibodymimic, a synthetic antibody, a single chain antibody or an equivalentthat binds to or complexes with a single biomarker.
 6. The diagnosticreagent according to claim 1, wherein any of said ligands comprises anucleotide sequence capable of hybridizing to a nucleic acid sequenceencoding a single biomarker.
 7. The diagnostic reagent according toclaim 1, wherein said substrate is a microarray, a microfluidics card, achip, a bead, or a chamber.
 8. A diagnostic reagent consisting of: (a) aligand capable of specifically complexing with, binding to, orquantitatively detecting or identifying the biomarker chlorideintracellular channel protein 4 (CLIC4) or an isoform, pro-form,modified molecular form, posttranslational modification, or uniquepeptide fragment or unique nucleic acid fragment thereof; (b) a ligandcapable of specifically complexing with, binding to, or quantitativelydetecting or identifying the biomarker chloride intracellular channelprotein 1 (CLIC1) or an isoform, pro-form, modified molecular form,posttranslational modification, or unique peptide fragment or uniquenucleic acid fragment thereof; and (c) a ligand capable of specificallycomplexing with, binding to, or quantitatively detecting or identifyingthe biomarker cathepsin D-30 (CTSD-30) or an isoform, pro-form, modifiedmolecular form, posttranslational modification, or unique peptidefragment or unique nucleic acid fragment thereof; and one or moreadditional ligands, each additional ligand capable of specificallycomplexing with, binding to, or quantitatively detecting, or identifyingan additional biomarker that indicates the presence of ovarian cancer ina human subject, wherein said additional biomarker is selected from: i.tropomyosin 1 (TPM1); ii. tropomyosin 2 (TPM2); iii. tropomyosin 3(TPM3); iv. tropomyosin 4 (TPM4); v. proteasome subunit alpha type-7(PSMA7); vi. CA125; and vii. HE4; wherein at least one of the ligands isattached to a detectable label or immobilized on a substrate.
 9. Thediagnostic reagent or device according to claim 8, wherein an isoform ofTPM1 is TPM1, variant 6 (UniProt ID No. Q1ZYL5) or TPM1, variant 8(UniProt ID No. B7Z596); wherein an isoform of TPM2 is TPM2 beta chain(UniProt ID No.UR1H P07951, UR1H₁₃Q5TCU3, or UR1H₁₃Q5TCU8) or TPM2,isoform 2 (UniProt ID No. P07951-2, A7XZE4), wherein an isoform of TPM3is TPM3 alpha-3 chain (UniProt ID No. P06753, Q5VU59, Q5HYB6, B2RDE1, orE2RB38); or wherein an isoform of TPM4 is TPM4 alpha-4 chain (UniProt IDNo. P6736), or TPM4, Isoform 2 (UniProt ID No. P67936-2).
 10. Thediagnostic reagent or device according to claim 8, wherein theadditional biomarker is CA125, or an isoform, pro-form, modifiedmolecular form, or unique peptide fragment therefrom; or HE4, or anisoform, pro-form, modified molecular form, or unique peptide fragmenttherefrom.